Compositions and methods for molecular labeling

ABSTRACT

The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional applicationSer. No. 14/995,744, filed Jan. 14, 2016, which is a continuation ofU.S. Nonprovisional application Ser. No. 14/874,553, filed Oct. 5, 2015,which is a continuation of U.S. Nonprovisional application Ser. No.13/398,677, filed Feb. 16, 2012, which claims the benefit of U.S.Provisional Application Ser. No. 61/444,612, filed Feb. 18, 2011, andU.S. Provisional Application Ser. No. 61/476,714, filed Apr. 18, 2011.The above-referenced applications are incorporated by reference hereinin their entireties.

FIELD OF THE INVENTION

The present invention generally relates to methods and materials forbuilding barcode libraries and labeling target materials, such asindividual cells or molecules, with labels such as barcode-type andprobe-type labels.

BACKGROUND OF THE INVENTION

The analysis of nucleic acids and proteins is an essential element ofmolecular biology. The ability to detect, discriminate, and utilizegenetic and proteomic information allows sensitive and specificdiagnostics, as well as the development of treatments. Most genetic andproteomic analysis requires labeling for detection of the analytes ofinterest. For example, in sequencing applications, nucleotides added toa template strand during sequencing-by-synthesis typically are labeled,or are intended to generate a label, upon incorporation into the growingstrand. The presence of the label allows detection of the incorporatednucleotide. Effective labeling techniques are desirable in order toimprove diagnostic and therapeutic results.

SUMMARY OF THE INVENTION

The present invention generally provides products and methods forlabeling target material in a fluid compartment. In particular, theinvention provides fluid compartments such as droplets for thesequestration, isolation, labeling, detection, identification, andanalysis of target material. The invention further provides labels.Labels according to the invention include barcode-type labels andprobe-type labels.

Principles of the invention can be applied to analyze all or a portionof an entire genome, transcriptome, or proteome. Techniques disclosedherein provide labeled materials isolated in fluid compartments for usewith analytical techniques such as sequencing, haplotyping, andmultiplex digital-PCR.

As disclosed herein, target material can be sequestered in a fluidcompartment or partition such as a single droplet. Other reagentsincluding labels (e.g., barcoded or optically-labeled N-mers) can beprovided, optionally also sequestered in droplets. The other reagentscan be introduced into the fluid partitions containing the targetmaterial, for example, by merging droplets, resulting in the labeling ofthe target molecules (e.g., by hybridization of N-mers to target nucleicacids). Target material can undergo optional processing such asselective enrichment, amplification, or capture on a substrate (e.g.,beads). Where the labels are of the barcode type, the invention providesanalytical methods including selective capture or enrichment,sequencing, haplotype phasing, genotyping, and improved sequence readassembly, as well as methods of producing barcode droplet libraries.Where the labels are of the probe-type, the invention provides noveldigital PCR assays including multiplex assays.

Target material can be obtained from a sample, and can include nucleicacid, proteins, carbohydrates, or other materials. The sample may be ahuman tissue or body fluid. Exemplary body fluids include pus, sputum,semen, urine, blood, saliva, and cerebrospinal fluid.

In certain aspects, the invention provides fluidic compartments tocontain all or a portion of a target material. In some embodiments, acompartment is droplet. While reference is made to “droplets” throughoutthe specification, that term is used interchangeably with fluidcompartment and fluid partition unless otherwise indicated. A fluidcompartment can be a slug, an area on an array surface, a globule, or areaction chamber in a microfluidic device, such as for example, amicrofluidic device fabricated using multilayer soft lithography (e.g.,integrated fluidic circuits). Except where indicated otherwise,“droplet” is used for convenience and any fluid partition or compartmentmay be used.

A droplet according to the invention generally includes an amount of afirst sample fluid in a second carrier fluid. Any technique known in theart for forming droplets may be used with methods of the invention. Anexemplary method involves flowing a stream of the sample fluidcontaining the target material (e.g., nucleic acid template) such thatit intersects two opposing streams of flowing carrier fluid. The carrierfluid is immiscible with the sample fluid. Intersection of the samplefluid with the two opposing streams of flowing carrier fluid results inpartitioning of the sample fluid into individual sample dropletscontaining the target material.

The carrier fluid may be any fluid that is immiscible with the samplefluid. An exemplary carrier fluid is oil. In certain embodiments, thecarrier fluid includes a surfactant, such as a fluorosurfactant.

The same method may be applied to create individual droplets thatcontain other reagents such as labels or reagents for an amplificationreaction such as a polymerase chain reaction (PCR), or a non-PCR basedamplification reaction such as multi-strand displacement amplification,or other methods known to one of ordinary skill in the art. Suitablereagents for conducting PCR-based amplification reactions are known tothose of ordinary skill in the art and include, but are not limited to,DNA polymerases such as Taq polymerase, forward and reverse primers,deoxynucleotide triphosphates (dNTPs), and one or more buffers. Suitablereagents for conducing non-PCR amplification reactions include, forexample, a high fidelity enzyme such as Φ29. Alternatively, atransposase can be used.

Either the droplets containing the first fluid, the droplets containingthe second fluid, or both, may be formed and then stored in a libraryfor later merging, aspects of certain implementations of which aredescribed in U.S. Pub. 2010/0022414, hereby incorporated herein in itsentirety for all purposes.

Once formed, droplets containing the target material can be merged withdroplets containing other reagents. Merging can produce a set ofdroplets, each containing target and other reagents such as, in eachdroplet, a single nucleic acid template and heterogeneous mixture ofprimer pairs and probes. Merging can be accomplished, for example, inthe presence of an electric field. Moreover, it is not required thatboth fluids be in the form of droplets when merging takes places. Oneexemplary method for merging of fluid portions with droplets is taught,for example, in co-pending U.S. Patent Application No. 61/441,985 andSer. No. 13/371,222, the contents of each of which are incorporated byreference herein.

In certain embodiments, fluidic compartments are formed by providing oneor more of a first fluid partition (e.g., a droplet) comprising a targetmaterial and a second fluid (e.g., as a fluid stream or within droplets)comprising a plurality of nucleic acid constructs, each containing afunctional N-mer capable of hybridizing to a unique region of the targetmaterial, and a unique N-mer to label the target. The first and secondfluids are merged to form a droplet. Merging can be accomplished byapplication of an electric field to the two fluids. In certainembodiments, the second fluid additionally contains reagents forconducting an amplification reaction, such as a polymerase chainreaction or a multiple displacement amplification reaction. Optionally,the genetic material can be fragmented or sheared using methods wellknown to those of skill in the art, for example, prior to sequesteringinto droplets or hybridizing to N-mers.

In certain aspects, the invention provides a method of making a barcodelibrary including obtaining a plurality of nucleic acid constructs inwhich each construct includes a unique N-mer and a functional N-mer. Thefunctional N-mer can be a random N-mer, a PCR primer, a universalprimer, an antibody, a sticky end, or any other sequence. The method caninclude making M sets of a number N of fluid compartments eachcontaining one or more copies of a unique construct. The method cancreate barcode libraries of higher complexity by adding an additionalconstruct to each compartment in a set, and repeating that for each setto produce NXM compartments each containing a unique pair of constructs.The pairs can be hybridized or ligated to produce new constructs. Ineach construct in a barcode library, each unique N-mer can be adaptedfor identification by sequencing, probe hybridization, other methods, ora combination of methods.

In certain aspects, the invention provides a method for labeling targetmaterial comprising segregating each of a plurality of targets into afluid compartment and providing one or more copies of a construct thatis unique for each fluid compartment, in which each construct includes aunique N-mer and a functional N-mer. The method can include associatingeach target with a copies of a construct, for example, by hybridization.Optional steps of methods of the invention can include performing anamplification reaction to produce amplicons that each contain a copy ofthe construct; releasing the contents of fluid compartments into a bulkphase; performing a second amplification reaction on amplicons;sequencing products of the invention; and detecting products of theinvention by digital PCR. Higher levels of complexity (e.g., forarbitrary high levels of multiplex parallel analysis) can obtained byintroducing into each fluid partition one or more copies of anadditional construct (for example, that are unique to a specific portionof a target) and linking each additional construct to a copy of theconstruct unique to each fluid partition. Target material can beunlabeled when segregated into the fluid compartments.

In certain aspects, the invention provides a compartment containing allor a portion of a target material, and a plurality of constructsincluding unique N-mers and functional N-mers (e.g., capable ofhybridizing to a unique region of the target material). Examples oftarget material include but are not limited DNA, genomic DNA,chromosome(s), RNA, expressed RNA and/or protein molecules. In someembodiments, the target material includes a single cell segregated intoa fluid compartment. The cell can be lysed within the compartment, andthe lysate can be targeted for labeling. Lysate can include the geneticor proteomic material derived from the single cell (prokaryotic oreukaryotic) or a subset thereof (e.g., an entire genome, transcriptome,proteome, or a portion thereof). Droplets containing cells may be sortedaccording to a sorting operation prior to merging with the otherreagents (e.g., as a second set of droplets). The other reagents maycontain reagents or enzymes such as a detergent or a protease (e.g., aheat activatable protease) that facilitates the breaking open of thecell and release of the nucleic acids therein. Once the reagents areadded to the droplets containing the cells (for example, through dropletmerging) and the cells are lysed, primers can be hybridized to thetarget and then target (e.g., nucleic acid) can be amplified, forexample, by PCR.

In certain embodiments, the invention provides a plurality of nucleicconstructs including a functional N-mer that comprises a randomsequence, for example, a 6-mer for use in a multiple displacementreaction (MDA). Alternatively, the N-mers can comprise a target specificsequence, such as a sequence specific for a gene, a gene mutation, agene motif, a splice site, a regulatory region of a gene, or a singlenucleotide polymorphism. In some embodiments, the N-mers can correspondto one or more consensus sequences, such as, for example, CPG motifs, orother sequence motifs that are related to known or suspected sequencesindicative of splice sites, promoter regions, regulatory regions, orother functional genomic units, etc. The N-mers can each furthercomprise a common sequence, such as a universal primer sequence. Incertain embodiments, the N-mers comprise oligo-dT labeled primers.

The invention generally provides methods and materials for labeling atarget material (e.g., protein or nucleic acid). Labeling can involvebarcode-type labeling using nucleic acid constructs or a probe-typelabel (e.g., for digital PCR). Nucleic acid constructs can involveinformational (i.e, unique or of known sequence) or functional N-mers.In certain embodiments, one or more constructs contain different uniqueN-mers (i.e., unique labels). The label is preferably associated with a5′ end of the N-mers. However, the label can be associated with a 3′ endof the N-mers.

The label associated with each of the N-mers can be a nucleic acid tag,or “barcode” sequence. Where a barcode is included, the N-mer generallyhybridizes to the target material and is copied throughout subsequentsteps such that the barcode is included in amplicons or sequence readsthat may result. Where a probe-type label is included, the N-mergenerally hybridizes to a specific material, for example, PCR productcontaining the target region, and can be detected in assays such asdigital PCR. A probe-type label can include an optical label such as afluorescent label. In some embodiments, an optical label is attached toan antibody specific for a target region of interest in a targetmaterial. Applications involving probe-type or barcode-type labels willbe discussed in greater detail below.

Whatever construct is used, a target material can be labeled by mergingdroplets containing the target material with a fluid stream or dropletstream containing the desired construct or merging a fluid stream of thetarget material with the construct into droplets.

The methods of the invention can further include the step of amplifyingor copying the target material so as to preserve, for each amplifiedproduct, an association between the amplified product and the label. Incertain aspects of the invention, the amplified product is indicative ofa haplotype. The nucleic acid template in each of the merged/formeddroplets is amplified, e.g., by thermocycling the droplets undertemperatures/conditions sufficient to conduct a PCR reaction. Theresulting amplicons in the droplets can then be analyzed. For example,using probe-type labels, the presence or absence of the plurality oftargets in the one or more droplets is detected optically, e.g., by thedetectable label on the plurality of probes. Alternatively, ampliconscan be sequenced and reads assembled based on the presence ofbarcode-type labels.

In some embodiments, capture sequences are introduced into dropletscontaining target material, for example, by merging the droplets with asecond set of droplets containing the capture sequences. Capturesequences can include a barcode label and a portion that is capable ofbeing captured on a solid surface (e.g., biotin/streptavidin on asurface; antibody/antigen; aptamers; anchored oligonucleotides; etc.). Adroplet containing a nucleic acid can be merged with a second dropletcontaining the capture sequence, preferably with a tag (i.e., abarcode-type label). The capture sequence is allowed to hybridize to thetarget nucleic acid. The emulsion is then broken to release thehybridized capture sequence and target nucleic acid. The releasednucleic acid is then captured on a solid support allowing the removal ofelements such as cell debris, proteases and detergents that may inhibitsubsequent steps. The tag is then incorporated by replication of thecaptured nucleic acid using the capture sequence with the tag as aprimer. Replication can generate DNA from either DNA or RNA (cDNAsynthesis). This material can either be processed directly or amplifiedfurther using methods known in the field such as PCR or multi-stranddisplacement amplification.

The capture sequences can be synthesized directly onto the beads or beattached by such means as biotinylated sequences and streptavidin beads.The use of streptavidin beads and biotinylated sequences has theadvantage of allowing a generic bead to be used with new libraries ofbiotinylated capture sequences that can be assembled on demand. However,any method known in the art for attaching nucleic acid sequences tobeads can be utilized.

In certain embodiments, droplets containing target material may bemerged with droplets containing beads that are designed to capture thetarget. After capture, the emulsion (i.e., set of droplets) is brokenand the beads are used to purify the target away from other componentsof the reaction that may inhibit subsequent steps such as cell debris,proteases and detergents. Target (e.g., nucleic acid) can be captured onbeads by using random N-mers designed to capture all sequences. In someembodiments, N-mers that are designed to capture only portions of thetarget are attached to the beads. Where the N-mers include abarcode-type tag, the tag can be incorporated by replication of thecaptured nucleic acid using the capture sequence with the tag as aprimer. The replication can generate DNA from DNA or RNA (cDNAsynthesis). This material can either be processed directly or amplifiedfurther using methods known in the art such as PCR or multi-stranddisplacement amplification.

In certain embodiments, methods of the invention include enriching allor selected portions of a target material. N-mers can be provided thatfurther contain a common nucleotide sequence, such as a universal PCRsequence. In an exemplary embodiment, the enrichment step isaccomplished by incorporating an adapter onto the 5′ end of theamplified genetic material, such as a universal PCR primer sequence, andfurther amplifying the genetic material. Only those strands having alabel will be amplified, thereby enriching for the labeled geneticmaterial. Alternatively, enrichment of sequence specific labeled strandscan be achieved through amplification using a primer specific for theuniversal priming sequence incorporated into the labeled strand, and aprimer specific for a desired target sequence. An enrichment step can bespecific for target regions of interest in the genetic material, such asconsensus sequences like CPG motifs, or other sequence motifs that arerelated to known or suspected sequences indicative of splice sites,promoter regions, regulatory regions, poly-A tail etc. In someembodiments, a first portion of amplified product associated with thelabel is enriched relative to a second portion of amplified product notassociated with the label (e.g., through the inclusion of universalpriming sites with the label).

In certain embodiments, the invention provides long sequences fromshort-read sequencing technologies. A set of primers is used that istiled across the sequence of interest. Target nucleic acid is isolatedin fluid partitions (e.g., droplets). Optionally, a plurality of targetsare isolated in droplets and analyzed in parallel. For each droplet, aset of primers is provided in which each primer includes a labelsequence that is unique for the droplet. the target nucleic acid isamplified in each droplet, with the result that every amplicon strandincludes the label sequence at each end. In some embodiments, thedroplets are ruptured and the amplicons are sequenced in such a way thateach sequence read contains the target label sequence. Since the primerpairs were tiled to cover a long sequence, the reads can be assembledinto “long reads” covering the sequence. Because each read is associatedwith a unique starting molecule through the presence of the labelsequence, each “long read” that is produced from short read assemblywill correspond to a single molecule of template. Thus, the sequencereads can be mapped back to the targeted genome, transcriptome,proteome, or a portion thereof.

Suitable sequencing methods include, but are not limited to, sequencingby hybridization, sequencing by synthesis technology (e.g., HiSeq™ andSolexa™, Illumina), SMRT™ (Single Molecule Real Time) technology(Pacific Biosciences), true single molecule sequencing (e.g.,HeliScope™, Helicos Biosciences), massively parallel next generationsequencing (e.g., SOLiD™, Applied Biosciences; Solexa and HiSeq™,Illumina), massively parallel semiconductor sequencing (e.g., IonTorrent), and pyrosequencing technology (e.g., GS FLX and GS JuniorSystems, Roche/454).

In certain aspects, the invention provides a barcode library, which canbe, for example, a stable barcode library which can be stored (e.g., fora year or longer). A barcode library can comprise a plurality of fluidcompartments, each containing one or more copies of a unique construct,in which each construct includes a unique N-mer and a functional N-mer.For a universal barcode library of general applicability, eachfunctional N-mer may be a sticky end, capable of being associated withanother sticky end. Other functional N-mers can includesequence-specific primers; random N-mers; antibodies; probe targets; anduniversal primer sites. The fluid compartments can be water-in-oildroplets. The unique N-mer offers a barcode of information and cangenerally be between about 2 and 21 nucleotides in length, and optionallonger, e.g., up to 50, 100, or any length.

In certain aspects, the invention relates to methods for detecting oridentifying one or a plurality of targets in a biological sample usingdigital PCR in fluid partitions. Methods of the invention includelabeling target material with a probe-type label. A probe type label caninclude an optical label, and labeled target material can be identifiedor analyzed using digital PCR.

Target material can be labeled with any suitable probe-type label knownin the art. Probes may generally include sequences designed to hybridizeto a target of interest. Detection of hybridization can indicate thatthe target of interest is present. Hybridization can be detected, forexample, by including a fluorescent label on a probe structured so thatthe label is quenched unless hybridized to the intended target of theprobe. Quenched and unquenched probes can be detected optically.

One or a plurality of such probes can be provided in a fluid partition.Members of the plurality of probes can each include the same detectablelabel, or a different detectable label. The plurality of probes caninclude one or more groups of probes at varying concentrations. The oneor more groups of probes can include the same detectable label whichvaries in intensity upon detection, due to the varying probeconcentrations. The droplets of the invention can further contain one ormore reagents for conducting a polymerase chain reaction (e.g.,polymerase, dNTPs, primers, etc.), for example, to enable probes tohybridize to amplified product (i.e., amplicons).

In some embodiments, the invention provides microfluidic droplets formultiplex analysis. Each droplet can contain a plurality of probes thathybridize to amplicons produced in the droplets. Preferably, the dropletcontains two or more probes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14,16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 500, or moreprobes.

The ability to amplify and detect single nucleic acids in dropletsenables digital PCR, detection, counting, and differentiation amongnucleic acids, especially those present in heterogeneous samples. Thus,the invention applies to digital amplification techniques and, inspecific embodiments enables multiplex PCR in droplets. For example,multiplexing primers in droplets enables the simultaneous increase inthe number of PCR droplets while keeping the amount of input DNA thesame or lower and generate the same or greater amplicon yield. Thisresults in an overall increase in the amount of PCR positive dropletsand amplicon yield without the consumption of more DNA. In someembodiments, even though the number of PCR primer pairs per droplet isgreater than one, there is only one template molecule per droplet, andthus, in some implementations, there is only one primer pair per dropletthat is being utilized at one time. As such, the advantages of dropletPCR for eliminating bias from either allele specific PCR or competitionbetween different amplicons is maintained. However, as described belowin relation to detection of haplotypes, other implementationsadvantageously allow detection of multiple loci on a single templateusing multiple primer pairs, preferably designed to minimize bias.

In certain aspects, the invention provides methods of forming fluidpartitions including target and reagents for digital PCR in which themethods enable multiplex digital PCR at high “plexity” in fluidpartitions. In some embodiments, one or more droplets are formed, eachcontaining a single nucleic acid template and a heterogeneous mixture ofprimer pairs and probes, each specific for multiple target sites on thetemplate. For example, a first fluid (either continuous, ordiscontinuous as in droplets) containing a single nucleic acid template(DNA or RNA) is merged with a second fluid (also either continuous, ordiscontinuous as in droplets) containing a plurality of primer pairs anda plurality of probes, each specific for multiple target sites on thenucleic acid template, to form a droplet containing the single nucleicacid template and a heterogeneous mixture of primer pairs and probes.The second fluid can also contain reagents for conducting a PCRreaction, such as a polymerase and dNTPs. The droplet contents can beamplified (e.g., by thermocycling). The probes are hybridized to theamplicons and hybridization is optically detected.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameor similar parts throughout the different views. Also, the drawings arenot necessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention.

FIG. 1 is a flow chart of the depicting an example of a labeling methodaccording to the invention.

FIG. 2A shows a method of merging droplets.

FIG. 2B is a block diagram of droplets for merging.

FIG. 2C shows a method of sequestering material in droplets

FIG. 2D shows products of a labeling step

FIG. 2E shows a 10-mer (SEQ ID: 1) and a schematic for a 6-mer.

FIG. 3A is a schematic depicting an example of barcode labeled strandsin a droplet before sequencing (in droplet) and after sequencing (inbulk).

FIG. 3B is a schematic depicting an example of a labeled primer having auniversal priming site before incorporation into/onto a target nucleicacid and after incorporation into a target nucleic acid.

FIG. 4A depicts a droplet formation device.

FIG. 4B depicts a portion of the droplet formation device of FIG. 4A.

FIGS. 5A-B show a method of making a universal barcode library.

FIGS. 6A-B show six types of barcodes with sticky end components.

FIGS. 7A-B show a universal barcode droplet library with targetingprimers.

FIGS. 8A-B show a universal barcode droplet library.

FIGS. 9A-B show ligating sticky-ended universal barcodes to barcoded PCRprimers.

FIGS. 10A-B show an overall workflow for single molecule barcodedhaplotype phasing.

FIGS. 11A-C show is a schematic depicting the PCR details of theschematic depicted in FIG. 35A-B.

FIGS. 12A-B show up front processing for amplification-based singlemolecule haplotyping with universal PCR barcodes.

FIGS. 13A-B show barcode addition for amplification-based singlemolecule haplotyping with universal PCR barcodes.

FIGS. 14A-B show labeling and release for amplification-based singlemolecule haplotyping with universal PCR barcodes.

FIGS. 15A-B show processing for amplification-free haplotyping.

FIGS. 16A-B show barcoding in amplification-free haplotyping.

FIGS. 17A-B show amplification free haplotyping.

FIGS. 18A-B show a general workflow for single cell genomics.

FIGS. 19A-B show single cell genomics using barcoded primers.

FIGS. 20A-B show single cell genomics using a universal barcode library.

FIG. 21 shows using a random hexamer library with phi29.

FIG. 22 shows a barcoded random hexamer library.

FIGS. 23A-B are a schematic depicting various exemplary barcode schemesfor the generation of an barcoded mRNA primer droplet library.

FIGS. 24A-B show a sorted cell workflow for barcoding transcriptomesfrom single cells.

FIGS. 25A-B show a sorted cell workflow for barcoding transcriptomesfrom single cells using a barcode library in a detergent lysis buffer.

FIGS. 26A-B show a bead-in-droplet workflow for barcodingtranscriptomes.

FIGS. 27A-C show barcoding biomarkers.

FIGS. 28A-C show barcoding biomarkers on a per-cell basis.

FIGS. 29A-B show a step for single cell digital biomarker counting.

FIGS. 30A-B show a step for single cell digital biomarker counting.

FIGS. 31A-D show a step for single cell digital biomarker counting.

FIGS. 32A-C show motifs for linking and releasing barcodes. FIGS. 32A-Calso shows SEQ IDs: 2-11.

FIGS. 33A-B show a workflow for digital droplet proteomics usingbarcoded antibodies.

FIGS. 34A-B show barcoding a binder.

FIGS. 35A-B are a schematic depicting an exemplary workflow of asandwich assay.

FIGS. 36A-B show use of a universal barcode library in a single celllysate sandwich assay.

FIGS. 37A-J show types of sandwich assays.

FIGS. 38A-B show use of universal barcodes in a binding partneridentification assay.

FIGS. 39A-B show barcodes for high-plex bead-based barcode labeling.

FIG. 40A-B depict a flowchart depicting the steps associated withisolation, encapsulation, molecular labeling, sorting and analysis ofsingle cell genomes using fluidic droplets.

FIG. 41 depicts lysis/proteolysis of cells (before and after) insidefluidic droplets.

FIG. 42 are images depicting the merger of droplets containing lysedcells and droplets containing reagents for WGA, and subsequent wholegenome amplification (WGA) in the merged fluidic droplets.

FIG. 43 shows high-accuracy next-generation sequencing (NGS).

FIG. 44 shows sequencing results.

FIG. 45 shows results from the 5× multiplexed droplet library FIG. 46shows results from a multiplexed copy number analysis.

FIG. 47 shows a detection apparatus according to certain embodiments.

FIG. 48A shows a droplet generation chip.

FIG. 48B depicts the droplet spacing for readout.

FIG. 48C depicts a cartoon of droplet readout by fluorescence.

FIGS. 49A-49C depict the serial dilution of template DNA quantified bydPCR.

FIG. 50A is a schematic representation of a droplet having 5 sets ofprimers for PCR amplification of a template sequence and 5 probes, eachlabeled with a fluorescent dye, that binds specifically to the amplifiedsequences.

FIG. 50B is a time trace of fluorescence intensity detected fromdroplets.

FIG. 50C is a scatter plot showing clusters corresponding to amplifiedsequences.

FIG. 51 is a schematic representation of a droplet having 5 sets ofprimers.

FIG. 52A is a scatter plot showing clusters representing amplifiedsequences.

FIG. 52B is a table showing the copy number of specific sequences.

FIGS. 53A-E schematically depict one-color detection of a geneticsequence with a microfluidic device.

FIGS. 54A-D show detection of two genetic sequences with a microfluidicdevice.

FIGS. 55A-D show detection of three genetic sequences with amicrofluidic device.

FIG. 56 shows dot plots depicting genetic sequences detected byfluorescence intensity.

FIG. 57A depicts a histogram of droplet peak fluorescence intensities.

FIG. 57B shows a comparison of gene copy numbers by monochromatic dPCR.

FIGS. 58A-C illustrate a schematic for tuning the intensity of adetectable label to a particular target with a microfluidic device.

FIG. 59 is a line graph depicting the linear dependence of dropletfluorescence intensity on probe concentration (Line, best linear fit(y=−0.092x+0.082, R²=0.995).

FIG. 60A is a 2D histogram of droplet fluorescence intensities.

FIG. 60B shows the results of the SMA pilot study.

FIG. 61 depicts a 9-plex dPCR assay for spinal muscular atrophy withonly two fluorophores, showing the process of optimizing dropletintensities.

FIG. 62 depicts an optical schematic for combining optical labels withmultiplexing.

FIG. 63 depicts a dPCR assay combining multiplexing with optical labelsusing co-flow microfluidic s.

FIGS. 64A-C show single assay selections using optical labels.

FIGS. 65A-C show single assay selections using optical labels.

FIGS. 66 A-C show single assay selections using optical labels.

FIGS. 67A-J depict a dPCR assay combining multiplexing with opticallabels.

FIG. 68 is a schematic showing haplotype detection in droplets.

FIGS. 69A-B show a workflow for restriction barcoding.

FIGS. 70A-B shows a workflow for barcoding sandwich assays for dPCRreadout.

FIGS. 71A-D shows droplet generation, merging, and combining

FIGS. 72A-D show droplet library generation and use in binding assays.

DETAILED DESCRIPTION

The invention generally provides materials and methods for labelingtarget nucleic acid, protein, or other material using microfluidicdroplet-based technology, and droplets produced using the same. Theinvention also provides the ability to associate sequencing reads withsingle cells in a heterogeneous mixture of cells. For example, one ormore mutations are identified in subpopulations of cancer cells in asample using the labeling methods of the invention. The ability toidentify multiple mutations existing in one cell better informs researchand physicians on the possibility of drug resistance or reoccurrence ofdisease, and also inform treatment. The invention further provides forthe ability to identify metagenomic loss of identity in individualbacteria (e.g., bacteria having multiple mutations in the same organismversus multiple bacteria, each having different mutations, in the samepopulation). In another aspect, the invention provides for the abilityto pool multiple patient samples in a multiplex sequencing reaction andto accurately identify the source of the multiple samples aftersequencing. Similarly, in proteomic assays (e.g., assays in which alabeled antibody or nucleic acid identifier (such as an aptamer) areused), methods of the invention provide accurate labeling and detection.

As discussed herein, the invention provides (I.) droplets for theanalysis and labeling of target material. The invention further provides(II.) barcode-type labels and (III.) probe-type labels.

I. Droplets

The invention provides microfluidic devices and systems for theformation of droplets and their manipulation (e.g., merging, sorting,rupturing, storing) for the analysis (e.g., amplification, labeling,detecting) of a variety of target materials FIG. 1 depicts a flow chartof the general methods of the invention. As shown in FIG. 1, the targetmaterial is encapsulated in a droplet, for example, using a microfluidicsystem. FIG. 2A-2C show droplet manipulation. FIG. 2D shows products ofa labeling step. FIG. 2E shows a 10-mer and a schematic for a 6-mer.FIG. 2C shows one exemplary method of sequestering material in droplets.Preferably, the genetic material is diluted such that each dropletcontains a single element (e.g., nucleic acid molecule, chromosome,genome, cell, protein, biological macromolecule, etc.). The elements canbe from a single cell (prokaryotic or eukaryotic), or a portion orsubset thereof (e.g., a single nucleic acid template). The droplets canoptionally be sorted (e.g., to identify subspecies that will besubsequently labeled). Where the genetic material is a single cell, thecells are lysed to release the genetic element in the single cell. Lysiscan be performed prior to encapsulation or after encapsulation (e.g.,using proteases, alkaline reagents, and/or detergents). Labels (e.g.,barcodes, fluorescent labels) can be introduced into the droplet andincorporated into or on the target. Optionally, an enrichment step canbe performed to enrich for the labeled genetic element, or sequencespecific enrichment.

Microfluidic Systems

Droplets can be generated using microfluidic systems or devices. As usedherein, the “micro-” prefix (for example, as “microchannel” or““microfluidic”), generally refers to elements or articles having widthsor diameters of less than about 1 mm, and less than about 100 microns(micrometers) in some cases. In some cases, the element or articleincludes a channel through which a fluid can flow. Additionally,“microfluidic”, as used herein, refers to a device, apparatus or systemthat includes at least one microscale channel.

Microfluidic systems and devices have been described in a variety ofcontexts, typically in the context of miniaturized laboratory (e.g.,clinical) analysis. Other uses have been described as well. For example,International Patent Application Publication Nos. WO 01/89788; WO2006/040551; WO 2006/040554; WO 2004/002627; WO 2008/063227; WO2004/091763; WO 2005/021151; WO 2006/096571; WO 2007/089541; WO2007/081385 and WO 2008/063227.

Specifically, the devices and methods described herein are based on thecreation and manipulation of aqueous phase droplets (e.g., dropletlibraries) surrounded by an immiscible carrier fluid. This combinationenables precise droplet generation, highly efficient, electricallyaddressable droplet coalescence, and controllable, electricallyaddressable single droplet sorting. Generally, microfluidic devicesinclude one or more channels in one or more analysis units. An “analysisunit” is a microsubstrate, e.g., a microchip. The terms microsubstrate,substrate, microchip, and chip are used interchangeably herein. Ananalysis unit typically includes at least an inlet channel and a mainchannel. The analysis unit can further include coalescence, detection,or sorting modules. The sorting module can be in fluid communicationwith branch channels which are in fluid communication with one or moreoutlet modules (e.g., collection module or waste module). For sortingapplications, at least one detection module cooperates with at least onesorting module to divert flow via a detector-originated signal. It shallbe appreciated that the “modules” and “channels” are in fluidcommunication with each other and therefore may overlap; i.e., there maybe no clear boundary where a module or channel begins or ends. Aplurality of analysis units of the invention may be combined in onedevice. The dimensions of the substrate are those of typical microchips,ranging between about 0.5 cm to about 15 cm per side and about 1 micronto about 1 cm in thickness. The analysis unit and specific modules aredescribed in further detail in WO 2006/040551; WO 2006/040554; WO2004/002627; WO 2004/091763; WO 2005/021151; WO 2006/096571; WO2007/089541; WO 2007/081385 and WO 2008/063227.

A variety of materials and methods can be used to form devices of theinvention. For example, components can be formed from solid materials,in which the channels can be formed via molding, micromachining, filmdeposition processes such as spin coating and chemical vapor deposition,laser fabrication, photolithographic techniques, etching methodsincluding wet chemical or plasma processes, and the like. See, forexample, Angell, et al., Scientific American, 248:44-55, 1983. At leasta portion of the fluidic system can be formed of silicone by molding asilicon chip. Devices of the invention can also be formed of a polymer,for example, an elastomeric polymer such as poly-dimethylsiloxane(PDMS), polytetrafluoroethylene (PTFE), Teflon®, or the like. PDMSpolymers include those sold under the trademark Sylgard by Dow ChemicalCo., Midland, Mich., and particularly Sylgard 182, Sylgard 184, andSylgard 186. Silicone polymers such as PDMS are generally inexpensive,readily available, and can be solidified from a prepolymeric liquid viacuring with heat. For example, PDMS is typically curable by exposure ofthe prepolymeric liquid to temperatures of about, for example, about 65°C. to about 75° C. for exposure times of, for example, about an hour.Also, silicone polymers can be elastomeric and thus may be useful forforming very small features with relatively high aspect ratios,necessary in certain embodiments of the invention.

Because PDMS can be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, devices of the inventionmay contain, at their surface, chemical groups capable of cross-linkingto other oxidized silicone polymer surfaces or to the oxidized surfacesof a variety of other polymeric and non-polymeric materials. Thus,components can be formed and then oxidized and essentially irreversiblysealed to other silicone polymer surfaces or to the surfaces without theneed for separate adhesives or other sealing means. In most cases,sealing can be completed simply by contacting an oxidized siliconesurface to another surface without the need to apply auxiliary pressureto form the seal. That is, the pre-oxidized silicone surface acts as acontact adhesive against suitable mating surfaces. Further, PDMS canalso be sealed irreversibly to a range of oxidized materials other thanitself including, for example, glass, silicon, silicon oxide, quartz,silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxypolymers, which have been oxidized in a similar fashion to the PDMSsurface (for example, via exposure to an oxygen-containing plasma).Molding, oxidation and sealing methods are described in the art, forexample, in Duffy et al., “Rapid Prototyping of Microfluidic Systems andPolydimethylsiloxane,” Anal. Chem., 70:474-480, 1998.

Another advantage of oxidized silicone polymers is that these surfacescan be much more hydrophilic than the surfaces of typical elastomericpolymers (where a hydrophilic interior surface is desired).

Thus, a channel can have a hydrophilic surface, which can be more easilywetted compared to other surfaces, which makes the channel easier tofill with aqueous solutions Generally, “channel,” as used herein, meansa feature on or in a substrate that at least partially directs the flowof a fluid. In some cases, the channel may be formed, at least in part,by a single component, e.g., an etched substrate or molded unit. Thechannel can have any cross-sectional shape, for example, circular, oval,triangular, irregular, square or rectangular (having any aspect ratio),or the like, and can be covered or uncovered (i.e., open to the externalenvironment surrounding the channel). In embodiments where the channelis completely covered, at least one portion of the channel can have across-section that is completely enclosed, and/or the entire channel maybe completely enclosed along its entire length with the exception of itsinlet and outlet. A channel can be formed, for example by etching asilicon chip using conventional photolithography techniques, or using amicromachining technology called “soft lithography” as described byWhitesides and Xia, Angewandte Chemie International Edition 37, 550(1998).

A fluid within a channel may partially or completely fill the channel.In some cases the fluid may be held or confined within the channel or aportion of the channel in some fashion, for example, using surfacetension (e.g., such that the fluid is held within the channel within ameniscus, such as a concave or convex meniscus). In an article orsubstrate, some (or all) of the channels may be of a particular size orless, for example, having a largest dimension perpendicular to fluidflow of less than about 5 mm, less than about 2 mm, less than about 1mm, less than about 500 microns, less than about 200 microns, less thanabout 100 microns, less than about 60 microns, less than about 50microns, less than about 40 microns, less than about 30 microns, lessthan about 25 microns, less than about 10 microns, less than about 3microns, less than about 1 micron, less than about 300 nm, less thanabout 100 nm, less than about 30 nm, or less than about 10 nm or less insome cases.

Channels can be configured to coalesce droplets or to flow material by adetection module or a sorting module. A main channel is typically influid communication with any coalescence, detection and/or sortingmodules, as well as inlet, branch, or outlet channels and any collectionor waste modules. These channels permit the flow of molecules, cells,small molecules or particles out of the main channel. An “inlet channel”permits the flow of molecules, cells, small molecules or particles intothe main channel. One or more inlet channels communicate with one ormore means for introducing a sample into the device of the presentinvention. A microfluidic device can also include fluid channels toinject or remove fluid in between droplets in a droplet stream for thepurpose of changing the spacing between droplets.

A microfluidic substrate can also include a specific geometry designedto prevent the aggregation of material prior to encapsulation indroplets. The geometry of channel dimension can be changed to disturbthe aggregates and break them apart by various methods, that caninclude, but is not limited to, geometric pinching (to force cells orparticles through a narrow region, whose dimension is smaller orcomparable to the dimension of a single cell) or a barricade (place aseries of barricades on the way of the moving cells to disturb themovement and break up the aggregates of cells).

To prevent target material (e.g., cells, molecules, or other material asdiscussed below) from adhering to the sides of the channels, thechannels (and coverslip, if used) may have a coating to minimizeadhesion. The surface of the channels can be coated with anyanti-wetting or blocking agent for the dispersed phase. The channel canbe coated with any protein to prevent adhesion of thebiological/chemical sample. Channels can be coated by any means known inthe art. For example, the channels can be coated with Teflon®, BSA,PEG-silane and/or fluorosilane in an amount sufficient to preventattachment and prevent clogging. In another example, the channels can becoated with a cyclized transparent optical polymer obtained bycopolymerization of perfluoro (alkenyl vinyl ethers), such as the typesold by Asahi Glass Co. under the trademark Cytop. In such an example,the coating is applied from a 0.1-0.5 wt % solution of Cytop CTL-809M inCT-Solv 180. This solution can be injected into the channels of amicrofluidic device via a plastic syringe. The device can then be heatedto about 90° C. for 2 hours, followed by heating at 200° C. for anadditional 2 hours. In another embodiment, the channels can be coatedwith a hydrophobic coating of perfluoro-alkylalkylsilane, described inU.S. Pat. No. 5,523,162. The surface of the channels in the microfluidicdevice can be also fluorinated by any means known in the art to preventundesired wetting behaviors. For example, a microfluidic device can beplaced in a polycarbonate dessicator with an open bottle of(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. The dessicatoris evacuated for 5 minutes, and then sealed for 20-40 minutes. Thedessicator is then backfilled with air and removed. This approach uses asimple diffusion mechanism to enable facile infiltration of channels ofthe microfluidic device with the fluorosilane and can be readily scaledup for simultaneous device fluorination. By fluorinating the surfaces ofthe channels, the continuous phase preferentially wets the channels andallows for the stable generation and movement of droplets through thedevice. The low surface tension of the channel walls thereby minimizesthe accumulation of channel clogging particulates, enhancing theprocessing of target material.

Target Material

Target materials for labeling, analysis, or detection according to themethods of the invention include, but are not limited to, cells, nucleicacids, proteins, multi-component complexes such as nucleic acid withassociated proteins (e.g., histones), chromosomes, carbohydrates, orsimilar materials. Methods of the invention are applicable to wholecells or to portions of genetic or proteomic material obtained fromcells. Target material generally includes anything that can besequestered into a fluid partition (e.g., droplet) and labeled.

Nucleic acid molecules include deoxyribonucleic acid (DNA) and/orribonucleic acid (RNA). Nucleic acid molecules can be synthetic orderived from naturally occurring sources. In one embodiment, nucleicacid molecules are isolated from a biological sample containing avariety of other components, such as proteins, lipids and non-templatenucleic acids. Nucleic acid template molecules can be obtained from anycellular material, obtained from an animal, plant, bacterium, fungus, orany other cellular organism. In certain embodiments, the nucleic acidmolecules are obtained from a single cell. Biological samples for use inthe present invention include viral particles or preparations. Nucleicacid molecules can be obtained directly from an organism or from abiological sample obtained from an organism, e.g., from blood, urine,cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue.Any tissue or body fluid specimen may be used as a source for nucleicacid for use in the invention. Nucleic acid molecules can also beisolated from cultured cells, such as a primary cell culture or a cellline. The cells or tissues from which template nucleic acids areobtained can be infected with a virus or other intracellular pathogen.

A sample can also be total RNA extracted from a biological specimen, acDNA library, viral, or genomic DNA. In certain embodiments, the nucleicacid molecules are bound as to other target molecules such as proteins,enzymes, substrates, antibodies, binding agents, beads, small molecules,peptides, or any other molecule and serve as a surrogate for quantifyingand/or detecting the target molecule. Generally, nucleic acid can beextracted from a biological sample by a variety of techniques such asthose described by Sambrook and Russell, Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor, N.Y. (2001). Nucleic acidmolecules may be single-stranded, double-stranded, or double-strandedwith single-stranded regions (for example, stem- and loop-structures).

Proteins or portions of proteins (amino acid polymers) that can bind tohigh affinity binding moieties, such as antibodies or aptamers, aretarget molecules for oligonucleotide labeling, for example, in droplets,in some embodiments of this invention.

Droplet Formation

Methods of the invention involve forming droplets, which may contain notarget material, target material from a single cell (e.g., a nucleicacid such as genomic DNA or expressed RNA), all or a portion of a targetfrom a single cell, or all or a portion of target from multiple cells(corresponding to limiting or terminal dilution, respectively, asdefined above).

In certain embodiments, the distribution of material within dropletsobeys the Poisson distribution. However, methods for non-Poisson loadingof droplets are known to those familiar with the art, and include butare not limited to active sorting of droplets, such as by laser-inducedfluorescence, or by passive one-to-one loading.

The droplets are aqueous droplets that are surrounded by an immisciblecarrier fluid. Methods of forming such droplets are discussed in U.S.Pub. 2008/0014589; U.S. Pub. 2008/0003142; U.S. Pub. 2010/0137163; U.S.Pat. No. 7,708,949; U.S. Pub. 2010/0172803; and U.S. Pat. No. 7,041,481,the content of each of which is incorporated by reference herein in itsentirety.

FIG. 4A shows an exemplary embodiment of a device 100 for dropletformation. Device 100 includes an inlet channel 101, and outlet channel102, and two carrier fluid channels 103 and 104. Channels 101, 102, 103,and 104 meet at a junction 105. Inlet channel 101 flows sample fluid tothe junction 105. Carrier fluid channels 103 and 104 flow a carrierfluid that is immiscible with the sample fluid to the junction 105.Inlet channel 101 narrows at its distal portion wherein it connects tojunction 105 (See FIG. 4B). Inlet channel 101 is oriented to beperpendicular to carrier fluid channels 103 and 104. Droplets are formedas sample fluid flows from inlet channel 101 to junction 105, where thesample fluid interacts with flowing carrier fluid provided to thejunction 105 by carrier fluid channels 103 and 104. Outlet channel 102receives the droplets of sample fluid surrounded by carrier fluid.

The sample fluid is typically an aqueous buffer solution, such asultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example bycolumn chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer,phosphate buffer saline (PBS) or acetate buffer. Any liquid or bufferthat is physiologically compatible with target material can be used. Thecarrier fluid is immiscible with the sample fluid. The carrier fluid canbe a non-polar solvent, decane (e g., tetradecane or hexadecane),fluorocarbon oil, silicone oil or another oil (e.g., mineral oil).

In certain embodiments, the carrier fluid contains one or moreadditives, such as agents which increase, reduce, or otherwise createnon-Newtonian surface tensions (surfactants) and/or stabilize dropletsagainst spontaneous coalescence on contact. Surfactants can includeTween, Span, fluorosurfactants, and other agents that are soluble in oilrelative to water. Suitable surfactants are known in the art. In someapplications, performance is improved by adding a second surfactant, orother agent, such as a polymer or other additive, to the sample fluid.Surfactants can aid in controlling or optimizing droplet size, flow anduniformity, for example by reducing the shear force needed to extrude orinject droplets into an intersecting channel. This can affect dropletvolume and periodicity, or the rate or frequency at which droplets breakoff into an intersecting channel. Furthermore, the surfactant can serveto stabilize aqueous emulsions in fluorinated oils from coalescing.

In certain embodiments, the droplets may be coated with a surfactant ora mixture of surfactants. In certain embodiments, the carrier fluid maybe caused to flow through the outlet channel so that the surfactant inthe carrier fluid coats the channel walls. In one embodiment, thefluorosurfactant can be prepared by reacting the perflourinatedpolyether DuPont Krytox 157 FSL, FSM, or FSH with ammonium hydroxide ina fluorinated solvent. The solvent, water, and ammonia can be removedwith a rotary evaporator. The surfactant can then be dissolved (e.g.,2.5 wt %) in a fluorinated oil (e.g., Flourinert (3M)), which thenserves as the carrier fluid.

Another approach to merging sample fluids involves forming a droplet,and contacting the droplet with a fluid stream, in which a portion ofthe fluid stream integrates with the droplet to form a mixed droplet.

A droplet is formed as described above. After formation of the sampledroplet from the first sample fluid, the droplet is contacted with aflow of a second sample fluid stream. Contact between the droplet andthe fluid stream results in a portion of the fluid stream integratingwith the droplet to form a mixed droplet, which, as discussed below,form a basis for droplet libraries according to certain embodiments ofthe invention.

The monodisperse droplets of the first sample fluid flow through a firstchannel separated from each other by immiscible carrier fluid andsuspended in the immiscible carrier fluid. The droplets are delivered tothe merge area, i.e., junction of the first channel with the secondchannel, by a pressure-driven flow generated by a positive displacementpump. While droplet arrives at the merge area, a bolus of a secondsample fluid is protruding from an opening of the second channel intothe first channel. Preferably, the channels are oriented perpendicularto each other. However, any angle that results in an intersection of thechannels may be used.

The bolus of the second sample fluid stream continues to increase insize due to pumping action of a positive displacement pump connected tochannel, which outputs a steady stream of the second sample fluid intothe merge area. The flowing droplet containing the first sample fluideventually contacts the bolus of the second sample fluid that isprotruding into the first channel. Contact between the two sample fluidsresults in a portion of the second sample fluid being segmented from thesecond sample fluid stream and joining with the first sample fluiddroplet to form a mixed droplet. In certain embodiments, each incomingdroplet of first sample fluid is merged with the same amount of secondsample fluid.

In certain embodiments, an electric charge is applied to the first orsecond sample fluids. Applying electric charge is described in U.S. Pub.2007/0003442, the content of which is incorporated by reference hereinin its entirety. Electric charge may be created in a sample fluid withinthe carrier fluid using any suitable technique, for example, by placingthe first and second sample fluids within an electric field (which maybe AC, DC, etc.), and/or causing a reaction to occur that causes thefirst and second sample fluids to have an electric charge, for example,a chemical reaction, an ionic reaction, a photocatalyzed reaction, etc.

The electric field, in some embodiments, is generated from an electricfield generator, i.e., a device or system able to create an electricfield that can be applied to the fluid. The electric field generator mayproduce an AC field (i.e., one that varies periodically with respect totime, for example, sinusoidally, sawtooth, square, etc.), a DC field(i.e., one that is constant with respect to time), a pulsed field, etc.The electric field generator may be constructed and arranged to createan electric field within a fluid contained within a channel or amicrofluidic channel. The electric field generator may be integral to orseparate from the fluidic system containing the channel or microfluidicchannel, according to some embodiments.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof. In some cases, transparent or substantiallytransparent electrodes can be used.

The electric field facilitates rupture of the interface separating thesecond sample fluid and the droplet. Rupturing the interface facilitatesmerging of a bolus of the second sample fluid and the first sample fluiddroplet. The forming mixed droplet continues to increase in size untilbreaks free from the second sample fluid stream, for instance prior tothe arrival of the next droplet containing the first sample fluid. Thesegmenting of the portion of the second sample fluid from the secondsample fluid stream occurs as soon as the shear force exerted on theforming mixed droplet by the immiscible carrier fluid overcomes thesurface tension whose action is to keep the segmenting portion of thesecond sample fluid connected with the second sample fluid stream. Thenow fully formed mixed droplet continues to flow through the firstchannel (e.g., for possible use in a droplet library).

Where material in droplets will be subject to PCR, those droplets can bemerged with a second fluid containing reagents for a PCR reaction (e.g.,Taq polymerase, dNTPs, magnesium chloride, and forward and reverseprimers, all suspended within an aqueous buffer). The second fluid mayalso include detectably labeled probes and/or universal barcodes fordetection of the amplified target material, the details of which arediscussed below. A droplet containing the target or portion thereof isthen caused to merge with the PCR reagents in the second fluid asdescribed above, producing a droplet that includes target and PCRreagents as well as, optionally, detectably labeled probes.

Droplet Libraries

Droplet libraries are useful to perform large numbers of assays whileconsuming only limited amounts of reagents. A “droplet,” as used herein,is an isolated portion of a first fluid that is surrounded by a secondfluid. In some cases, the droplets may be spherical or substantiallyspherical; however, in other cases, the droplets may be non-spherical,for example, the droplets may have the appearance of “blobs” or otherirregular shapes, for instance, depending on the external environment.In some embodiments, a droplet is a first fluid completely surrounded bya second fluid. As used herein, a first entity is “surrounded” by asecond entity if a closed loop can be drawn or idealized around thefirst entity through only the second entity (with the sometimesexception for portions of the first fluid that may be in contact with awall or other boundary, where applicable).

In general, a droplet library is made up of a number of library elementsthat are pooled together in a single collection. Libraries may vary incomplexity from a single library element to 10¹⁵ library elements ormore. Each library element is one or more given components at a fixedconcentration. The element may be, but is not limited to, cells, virus,bacteria, yeast, beads, amino acids, proteins, polypeptides, nucleicacids, polynucleotides or small molecule chemical compounds. The elementmay contain an identifier such as a label. The terms “droplet library”or “droplet libraries” are also referred to herein as an “emulsionlibrary” or “emulsion libraries.” These terms are used interchangeablythroughout the specification.

A cell library element can include, but is not limited to, hybridomas,B-cells, primary cells, cultured cell lines, cancer cells, stem cells,or any other cell type. Cellular library elements are prepared byencapsulating a number of cells from one to tens of thousands inindividual droplets. The number of cells encapsulated is usually givenby Poisson statistics from the number density of cells and volume of thedroplet. However, in some cases the number deviates from Poissonstatistics as described in Edd et al., “Controlled encapsulation ofsingle-cells into monodisperse picolitre drops.” Lab Chip,8(8):1262-1264, 2008. The discreet nature of cells allows for librariesto be prepared in mass with a plurality of cellular variants all presentin a single starting media and then that media is broken up intoindividual droplet capsules that contain at most one cell. Theseindividual droplets capsules are then combined or pooled to form alibrary consisting of unique library elements. Cell division subsequentto, or in some embodiments following, encapsulation produces a clonallibrary element.

A bead based library element contains one or more beads, and may alsocontain other reagents, such as antibodies, enzymes or other proteins.In the case where all library elements contain different types of beads,but the same surrounding media, the library elements can all be preparedfrom a single starting fluid or have a variety of starting fluids. Inthe case of cellular libraries prepared in mass from a collection ofvariants, the library elements will be prepared from a variety ofstarting fluids.

Often it is desirable to have exactly one cell per droplet with only afew droplets containing more than one cell when starting with aplurality of cells. In some cases, variations from Poisson statisticscan be achieved to provide an enhanced loading of droplets such thatthere are more droplets with exactly one cell per droplet and fewexceptions of empty droplets or droplets containing more than one cell.

Examples of droplet libraries are collections of droplets that havedifferent contents, ranging from beads, cells, small molecules, DNA,primers, antibodies. The droplets range in size from roughly 0.5 micronto 500 micron in diameter, which corresponds to about 1 pico liter to 1nano liter. However, droplets can be as small as 5 microns and as largeas 500 microns. Preferably, the droplets are at less than 100 microns,about 1 micron to about 100 microns in diameter. The most preferred sizeis about 20 to 40 microns in diameter (10 to 100 picoliters). Thepreferred properties examined of droplet libraries include osmoticpressure balance, uniform size, and size ranges.

The droplets comprised within the droplet library provided by theinstant invention are preferably uniform in size. That is, the diameterof any droplet within the library will vary less than 5%, 4%, 3%, 2%, 1%or 0.5% when compared to the diameter of other droplets within the samelibrary. The uniform size of the droplets in the library is critical tomaintain the stability and integrity of the droplets and is alsoessential for the subsequent use of the droplets within the library forthe various biological and chemical assays described herein.

In certain embodiments, the droplet libraries are using an immisciblefluorocarbon oil. The oil can comprise at least one fluorosurfactant. Insome embodiments, the fluorosurfactant comprised within immisciblefluorocarbon oil is a block copolymer consisting of one or moreperfluorinated polyether (PFPE) blocks and one or more polyethyleneglycol (PEG) blocks. In other embodiments, the fluorosurfactant is atriblock copolymer consisting of a PEG center block covalently bound totwo PFPE blocks by amide linking groups. The presence of thefluorosurfactant (similar to uniform size of the droplets in thelibrary) is critical to maintain the stability and integrity of thedroplets and is also essential for the subsequent use of the dropletswithin the library for the various biological and chemical assaysdescribed herein. Fluids (e.g., aqueous fluids, immiscible oils, etc.)and other surfactants that can be utilized in the droplet libraries ofthe present invention are described in greater detail herein.

The droplet libraries of the present invention are very stable and arecapable of long-term storage. The droplet libraries are determined to bestable if the droplets comprised within the libraries maintain theirstructural integrity, that is the droplets do not rupture and elementsdo not diffuse from the droplets. The droplets libraries are alsodetermined to be stable if the droplets comprised within the librariesdo not coalesce spontaneously (without additional energy input, such aselectrical fields described in detail herein). Stability can be measuredat any temperature. For example, the droplets are very stable and arecapable of long-term storage at any temperature; for example, e.g., −70°C., 0° C., 4° C., 37° C., room temperature, 75° C. and 95° C.Specifically, the droplet libraries of the present invention are stablefor at least 30 days. More preferably, the droplets are stable for atleast 60 days. Most preferably, the droplets are stable for at least 90days.

The invention provides a droplet library comprising a plurality ofaqueous droplets within an immiscible fluid (optionally comprising afluorosurfactant), wherein each droplet is preferably substantiallyuniform in size and comprises a different library element. The inventionprovides a method for forming the droplet library comprising providing asingle aqueous fluid comprising different library elements,encapsulating each library element into an aqueous droplet within animmiscible fluid (optionally comprising a fluorosurfactant).

In certain embodiments, different types of elements (e.g., cells orbeads), are pooled in a single source contained in the same medium.After the initial pooling, the elements are then encapsulated indroplets to generate a library of droplets wherein each droplet with adifferent type of bead or cell is a different library element. Thedilution of the initial solution enables the encapsulation process. Insome embodiments, the droplets formed will either contain a singleelement or will not contain anything, i.e., be empty. In otherembodiments, the droplets formed will contain multiple copies of alibrary element. The elements being encapsulated are generally variantsof a type. In one example, elements are cancer cells of a tissue biopsy,and each cell type is encapsulated to be screened for genomic data oragainst different drug therapies. Another example is that 10¹¹ or 10¹⁵different type of bacteria; each having a different plasmid splicedtherein, are encapsulated. One example is a bacterial library where eachlibrary element grows into a clonal population that secretes a varianton an enzyme.

In certain embodiments, a droplet library comprises a plurality ofaqueous droplets within an immiscible fluid, such that there is a singlemolecule contained within a droplet for every 20-60 droplets produced(e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60 droplets, or any integer inbetween). Single molecules are encapsulated by diluting the solutioncontaining the molecules to such a low concentration that theencapsulation of single molecules is enabled. In one specific example, aLacZ plasmid DNA was encapsulated at a concentration of 20 fM after twohours of incubation such that there was about one gene in 40 droplets,where 10 am droplets were made at 10 kHz per second. Formation of theselibraries relies on limiting dilutions.

The present invention also provides a droplet library comprising atleast a first aqueous droplet and at least a second aqueous dropletwithin a fluorocarbon oil comprising at least one fluorosurfactant,wherein the at least first and the at least second droplets are uniformin size and comprise a different aqueous fluid and a different libraryelement. The present invention also provides a method for forming theemulsion library comprising providing at least a first aqueous fluidcomprising at least a first library of elements, providing at least asecond aqueous fluid comprising at least a second library of elements,encapsulating each element of said at least first library into at leasta first aqueous droplet within an immiscible fluorocarbon oil comprisingat least one fluorosurfactant, encapsulating each element of said atleast second library into at least a second aqueous droplet within animmiscible fluorocarbon oil comprising at least one fluorosurfactant,wherein the at least first and the at least second droplets are uniformin size and comprise a different aqueous fluid and a different libraryelement, and pooling the at least first aqueous droplet and the at leastsecond aqueous droplet within an immiscible fluorocarbon oil comprisingat least one fluorosurfactant thereby forming an emulsion library.

For example, in one type of emulsion library, there are library elementsthat have different particles, i.e., cells or beads in a differentmedium and are encapsulated prior to pooling. In one example, aspecified number of library elements, i.e., n number of different cellsor beads, are contained within different mediums. Each of the libraryelements are separately emulsified and pooled, at which point each ofthe n number of pooled different library elements are combined andpooled into a single pool. The resultant pool contains a plurality ofwater-in-oil emulsion droplets each containing a different type ofparticle.

In some embodiments, the droplets formed will either contain a singlelibrary element or will not contain anything, i.e., be empty. In otherembodiments, the droplets formed will contain multiple copies of alibrary element. The contents of the beads follow a Poissondistribution, where there is a discrete probability distribution thatexpresses the probability of a number of events occurring in a fixedperiod of time if these events occur with a known average rate andindependently of the time since the last event. The oils and surfactantsused to create the libraries prevent the exchange of the contents of thelibrary between droplets.

FIG. 71A-D shows droplet generation, merging, and combining. As shown inpanel A, monodisperse aqueous droplets are formed in a fluorocarbon oilusing pressure-driven flow into a microfluidic nozzle. Panel B presentsa schematic showing surfactant “wall” which provides stability fordroplet manipulations. Panel C includes an image from a sequenceenrichment application, combining DNA samples with PCR reagents (mergingwith Primer Droplet Library). The mixed droplets can be taken off-chipfor PCR. In panel D, the top image shows ˜2×108 droplets before PCR, andbottom shows a microscope image of intact droplets after thermocycling.

FIG. 72A-D shows droplet library generation and use in binding assays(discussed in greater detail herein). As shown in Panel A, stabledroplet library reagents can be formulated (5-member library shown) andaliquoted for later use. Panel B shows reinjection of a droplet libraryand re-spacing into single file droplets. Panel C presents two bindingassays: microscope image on the top shows virus particles binding toembryonic fibroblasts; the image on the bottom shows individual beadsbinding to ELISA sandwich reagents in droplets.

Droplet Sorting

Methods of the invention may further include sorting the droplets basedupon whether the droplets contain a homogeneous population of moleculesor a heterogeneous population of molecules. A sorting module may be ajunction of a channel where the flow of droplets can change direction toenter one or more other channels, e.g., a branch channel, depending on asignal received in connection with a droplet interrogation in thedetection module. Typically, a sorting module is monitored and/or underthe control of the detection module, and therefore a sorting module maycorrespond to the detection module. The sorting region is incommunication with and is influenced by one or more sorting apparatuses.

A sorting apparatus includes techniques or control systems, e.g.,dielectric, electric, electro-osmotic, (micro-) valve, etc. A controlsystem can employ a variety of sorting techniques to change or directthe flow of molecules, cells, small molecules or particles into apredetermined branch channel. A branch channel is a channel that is incommunication with a sorting region and a main channel. The main channelcan communicate with two or more branch channels at the sorting moduleor branch point, forming, for example, a T-shape or a Y-shape. Othershapes and channel geometries may be used as desired. Typically, abranch channel receives droplets of interest as detected by thedetection module and sorted at the sorting module. A branch channel canhave an outlet module and/or terminate with a well or reservoir to allowcollection or disposal (collection module or waste module, respectively)of the molecules, cells, small molecules or particles. Alternatively, abranch channel may be in communication with other channels to permitadditional sorting.

A characteristic of a fluidic droplet may be sensed and/or determined insome fashion, for example, as described herein (e.g., fluorescence ofthe fluidic droplet may be determined), and, in response, an electricfield may be applied or removed from the fluidic droplet to direct thefluidic droplet to a particular region (e.g. a channel). In certainembodiments, a fluidic droplet is sorted or steered by inducing a dipolein the uncharged fluidic droplet (which may be initially charged oruncharged), and sorting or steering the droplet using an appliedelectric field. The electric field may be an AC field, a DC field, etc.For example, a channel containing fluidic droplets and carrier fluid,divides into first and second channels at a branch point. Generally, thefluidic droplet is uncharged. After the branch point, a first electrodeis positioned near the first channel, and a second electrode ispositioned near the second channel. A third electrode is positioned nearthe branch point of the first and second channels. A dipole is theninduced in the fluidic droplet using a combination of the electrodes.The combination of electrodes used determines which channel will receivethe flowing droplet. Thus, by applying the proper electric field, thedroplets can be directed to either the first or second channel asdesired. Further description of droplet sorting is shown in U.S. Pub.2008/0014589; U.S. Pub. 2008/0003142, and U.S. Pub. 2010/0137163.

Based upon the detected signal at the detection module, dropletscontaining a heterogeneous population of molecules are sorted away fromdroplets that contain a homogeneous population of molecules. Dropletsmay be further sorted to separate droplets that contain a homogeneouspopulation of amplicons of the target from droplets that contain ahomogeneous population of amplicons of the variant of the target.

Target Amplification

Methods of the invention may further involve amplifying the targetgenetic material in each droplet. Amplification refers to production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction or other technologies well known inthe art (e.g., Dieffenbach and Dveksler, PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y. [1995]). Theamplification reaction may be any amplification reaction known in theart that amplifies nucleic acid molecules, such as polymerase chainreaction, nested polymerase chain reaction, ligase chain reaction(Barany F. (1991) PNAS 88:189-193; Barany F. (1991) PCR Methods andApplications 1:5-16), ligase detection reaction (Barany F. (1991) PNAS88:189-193), strand displacement amplification, transcription basedamplification system, nucleic acid sequence-based amplification, rollingcircle amplification, and hyper-branched rolling circle amplification.

In certain embodiments, the amplification reaction is the polymerasechain reaction. Polymerase chain reaction (PCR) refers to methods by K.B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporatedby reference) for increasing concentration of a segment of a targetsequence in a mixture of genomic DNA without cloning or purification.The process for amplifying the target sequence includes introducing anexcess of oligonucleotide primers to a DNA mixture containing a desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The primers are complementary to theirrespective strands of the double stranded target sequence.

To effect amplification, primers are annealed to their complementarysequence within the target molecule. Following annealing, the primersare extended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one cycle; there can be numerous cycles) to obtaina high concentration of an amplified segment of a desired targetsequence. The length of the amplified segment of the desired targetsequence is determined by relative positions of the primers with respectto each other and by cycling parameters, and therefore, this length is acontrollable parameter.

The sample droplet may be pre-mixed with a primer or primers, or theprimer or primers may be added to the droplet. In some embodiments,droplets created by segmenting the starting sample are merged with asecond set of droplets including one or more primers for the targetnucleic acid in order to produce final droplets.

In embodiments involving merging of droplets, two droplet formationmodules are used. In one embodiment, a first droplet formation moduleproduces the sample droplets consistent with limiting or terminaldilution of target nucleic acid. A second droplet formation orreinjection module inserts droplets that contain reagents for a PCRreaction. Such droplets generally include the “PCR master mix” (known tothose in the art as a mixture containing at least Taq polymerase,deoxynucleotides of type A, C, G and T, and magnesium chloride) andforward and reverse primers (known to those in the art collectively as“primers”), all suspended within an aqueous buffer. The second dropletalso includes detectably labeled probes for detection of the amplifiedtarget nucleic acid, the details of which are discussed below. Differentarrangements of reagents between the two droplet types is envisioned.For example, in another embodiment, the template droplets also containthe PCR master mix, but the primers and probes remain in the seconddroplets. Any arrangement of reagents and template DNA can be usedaccording to the invention.

Primers can be prepared by a variety of methods including but notlimited to cloning of appropriate sequences and direct chemicalsynthesis using methods well known in the art (Narang et al., MethodsEnzymol., 68:90 (1979); Brown et al., Methods Enzymol., 68:109 (1979)).Primers can also be obtained from commercial sources such as OperonTechnologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies.The primers can have an identical melting temperature. The lengths ofthe primers can be extended or shortened at the 5′ end or the 3′ end toproduce primers with desired melting temperatures. Also, the annealingposition of each primer pair can be designed such that the sequence and,length of the primer pairs yield the desired melting temperature. Thesimplest equation for determining the melting temperature of primerssmaller than 25 base pairs is the Wallace Rule (Td=2(A+T)+4(G+C)).Another method for determining the melting temperature of primers is thenearest neighbor method (SantaLucia, “A unified view of polymer,dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics”,1998, P.N.A.S., 95 (4): 1460-5). Computer programs can also be used todesign primers, including but not limited to Array Designer Software(Arrayit Inc.), Oligonucleotide Probe Sequence Design Software forGenetic Analysis (Olympus Optical Co.), NetPrimer, and DNAsis fromHitachi Software Engineering. The TM (melting or annealing temperature)of each primer is calculated using software programs such as OligoDesign, available from Invitrogen Corp.

In one embodiment, the droplet formation modules are arranged andcontrolled to produce an interdigitation of sample droplets and PCRreagent droplets flowing through a channel. Such an arrangement isdescribed U.S. Pub. 2008/0014589; U.S. Pub. 2008/0003142, and U.S. Pub.2010/0137163.

A sample droplet is then caused to merge with a PCR reagent droplet,producing a droplet that includes the PCR master mix, primers,detectably labeled probes, and the target nucleic acid. Droplets may bemerged for example by: producing dielectrophoretic forces on thedroplets using electric field gradients and then controlling the forcesto cause the droplets to merge; producing droplets of different sizesthat thus travel at different velocities, which causes the droplets tomerge; and producing droplets having different viscosities that thustravel at different velocities, which causes the droplets to merge witheach other. Further discussion can be found in U.S. Pub. 2007/0003442.

In another embodiment, called simple droplet generation, a singledroplet formation module, or a plurality of droplet formation modulesare arranged to produce droplets from a mixture already containing thetemplate DNA, the PCR master mix, primers, and detectably labeledprobes. In yet another embodiment, called co-flow, upstream from asingle droplet formation module two channels intersect allowing two flowstreams to converge. One flow stream contains one set of reagents andthe template DNA, and the other contains the remaining reagents. In thepreferred embodiment for co-flow, the template DNA and the PCR mastermix are in one flow stream, and the primers and probes are in the other.On convergence of the flow streams in a fluidic intersection, the flowstreams may or may not mix before the droplet generation nozzle. Ineither embodiment, some amount of fluid from the first stream, and someamount of fluid from the second stream are encapsulated within a singledroplet. Following encapsulation, complete mixing occurs.

Once final droplets have been produced by any of the droplet formingembodiments above, or by any other embodiments, the droplets are thermalcycled, resulting in amplification of the target nucleic acid in eachdroplet. In certain embodiments, the droplets are collected off-chip asan emulsion in a PCR thermal cycling tube and then thermally cycled in aconventional thermal cycler. Temperature profiles for thermal cyclingcan be adjusted and optimized as with any conventional DNA amplificationby PCR.

In certain embodiments, the droplets are flowed through a channel in aserpentine path between heating and cooling lines to amplify the nucleicacid in the droplet. The width and depth of the channel may be adjustedto set the residence time at each temperature, which can be controlledto anywhere between less than a second and minutes.

In certain embodiments, the three temperature zones are used for theamplification reaction. The three temperature zones are controlled toresult in denaturation of double stranded nucleic acid (high temperaturezone), annealing of primers (low temperature zones), and amplificationof single stranded nucleic acid to produce double stranded nucleic acids(intermediate temperature zones). The temperatures within these zonesfall within ranges well known in the art for conducting PCR reactions.See for example, Sambrook et al. (Molecular Cloning, A LaboratoryManual, 3^(rd) edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001).

In certain embodiments, the three temperature zones are controlled tohave temperatures as follows: 95° C. (T_(H)), 55° C. (T_(L)), 72° C.(T_(M)). The prepared sample droplets flow through the channel at acontrolled rate. The sample droplets first pass the initial denaturationzone (T_(H)) before thermal cycling. The initial preheat is an extendedzone to ensure that nucleic acids within the sample droplet havedenatured successfully before thermal cycling. The requirement for apreheat zone and the length of denaturation time required is dependenton the chemistry being used in the reaction. The samples pass into thehigh temperature zone, of approximately 95° C., where the sample isfirst separated into single stranded DNA in a process calleddenaturation. The sample then flows to the low temperature, ofapproximately 55° C., where the hybridization process takes place,during which the primers anneal to the complementary sequences of thesample. Finally, as the sample flows through the third mediumtemperature, of approximately 72° C., the polymerase process occurs whenthe primers are extended along the single strand of DNA with athermostable enzyme. Methods for controlling the temperature in eachzone may include but are not limited to electrical resistance, peltierjunction, microwave radiation, and illumination with infrared radiation.

The nucleic acids undergo the same thermal cycling and chemical reactionas the droplets passes through each thermal cycle as they flow throughthe channel. The total number of cycles in the device is easily alteredby an extension of thermal zones or by the creation of a continuous loopstructure. The sample undergoes the same thermal cycling and chemicalreaction as it passes through N amplification cycles of the completethermal device.

In other embodiments, the temperature zones are controlled to achievetwo individual temperature zones for a PCR reaction. In certainembodiments, the two temperature zones are controlled to havetemperatures as follows: 95° C. (T_(H)) and 60° C. (T_(L)). The sampledroplet optionally flows through an initial preheat zone before enteringthermal cycling. The preheat zone may be important for some chemistryfor activation and also to ensure that double stranded nucleic acid inthe droplets are fully denatured before the thermal cycling reactionbegins. In an exemplary embodiment, the preheat dwell length results inapproximately 10 minutes preheat of the droplets at the highertemperature.

The sample droplet continues into the high temperature zone, ofapproximately 95° C., where the sample is first separated into singlestranded DNA in a process called denaturation. The sample then flowsthrough the device to the low temperature zone, of approximately 60° C.,where the hybridization process takes place, during which the primersanneal to the complementary sequences of the sample. Finally thepolymerase process occurs when the primers are extended along the singlestrand of DNA with a thermostable enzyme. The sample undergoes the samethermal cycling and chemical reaction as it passes through each thermalcycle of the complete device. The total number of cycles in the deviceis easily altered by an extension of block length and tubing.

In another embodiment the droplets are created and/or merged on chipfollowed by their storage either on the same chip or another chip or offchip in some type of storage vessel such as a PCR tube. The chip orstorage vessel containing the droplets is then cycled in its entirety toachieve the desired PCR heating and cooling cycles.

In another embodiment the droplets are collected in a chamber where thedensity difference between the droplets and the surrounding oil allowsfor the oil to be rapidly exchanged without removing the droplets. Thetemperature of the droplets can then be rapidly changed by exchange ofthe oil in the vessel for oil of a different temperature. This techniqueis broadly useful with two and three step temperature cycling or anyother sequence of temperatures.

Release from Droplet

Methods of the invention may further involve releasing amplified targetmolecules from the droplets for further analysis. Methods of releasingamplified target molecules from the droplets are shown in publicationsand patents referenced above.

In certain embodiments, sample droplets are allowed to cream to the topof the carrier fluid. By way of non-limiting example, the carrier fluidcan include a perfluorocarbon oil that can have one or more stabilizingsurfactants. The droplet rises to the top or separates from the carrierfluid by virtue of the density of the carrier fluid being greater thanthat of the aqueous phase that makes up the droplet. For example, theperfluorocarbon oil used in one embodiment of the methods of theinvention is 1.8, compared to the density of the aqueous phase of thedroplet, which is 1.0.

The creamed liquids are then placed onto a second carrier fluid whichcontains a de-stabilizing surfactant, such as a perfluorinated alcohol(e.g. 1H,1H,2H,2H-Perfluoro-1-octanol). The second carrier fluid canalso be a perfluorocarbon oil. Upon mixing, the aqueous droplets beginsto coalesce, and coalescence is completed by brief centrifugation at lowspeed (e.g., 1 minute at 2000 rpm in a microcentrifuge). The coalescedaqueous phase can now be removed and the further analyzed.

The released amplified material can also be subjected to furtheramplification by the use tailed primers and secondary PCR primers. Inthis embodiment the primers in the droplet contain an additionalsequence or tail added onto the 5′ end of the sequence specific portionof the primer. The sequences for the tailed regions are the same foreach primer pair and are incorporated onto the 5′ portion of theamplicons during PCR cycling. Once the amplicons are removed from thedroplets, another set of PCR primers that can hybridize to the tailregions of the amplicons can be used to amplify the products throughadditional rounds of PCR. The secondary primers can exactly match thetailed region in length and sequence or can themselves containadditional sequence at the 5′ ends of the tail portion of the primer.

During the secondary PCR cycling these additional regions also becomeincorporated into the amplicons. These additional sequences can include,but are not limited to: adaptor regions utilized by sequencing platformsfor library preparation; barcode sequences for the identification ofsamples multiplexed into the same reaction; molecules for the separationof amplicons from the rest of the reaction materials (e.g., biotin,digoxin, peptides, or antibodies); or molecules such as fluorescentmarkers that can be used to identify the fragments.

In certain embodiments, the amplified target molecules are sequenced. Ina particular embodiment, the sequencing is single-moleculesequencing-by-synthesis. Single-molecule sequencing is shown in U.S.Pat. Nos. 7,169,560; 6,818,395; 7,282,337; U.S. Pub. 2002/0164629; andBraslavsky, et al., PNAS (USA), 100: 3960-3964 (2003), the contents ofeach of these references are incorporated by reference herein in itsentirety.

Briefly, a single-stranded nucleic acid (e.g., DNA or cDNA) ishybridized to oligonucleotides attached to a surface of a flow cell. Thesingle-stranded nucleic acids may be captured by methods known in theart, such as those shown in U.S. Pat. No. 7,666,593. Theoligonucleotides may be covalently attached to the surface or variousattachments other than covalent linking as known to those of ordinaryskill in the art may be employed. Moreover, the attachment may beindirect, e.g., via the polymerases of the invention directly orindirectly attached to the surface. The surface may be planar orotherwise, and/or may be porous or non-porous, or any other type ofsurface known to those of ordinary skill to be suitable for attachment.The nucleic acid is then sequenced by imaging the polymerase-mediatedaddition of fluorescently-labeled nucleotides incorporated into thegrowing strand surface oligonucleotide, at single molecule resolution.

II. Barcode-Type Labels Barcode Sequences

The invention provides labels for target materials comprising adetectable barcode-type label. A detectable barcode-type label can beany barcode-type label known in the art including, for example,radio-frequency tags, semiconductor chips, barcoded magnetic beads(e.g., from Applied Biocode, Inc., Santa Fe Springs, Calif.), andnucleic acid sequences. In certain embodiments, a barcode-type label isa nucleic acid construct such as a nucleic acid construct including abarcode-type sequence (e.g., a unique N-mer). A construct of theinvention generally includes a functional portion. Thus, a barcodesequence generally refers to a nucleic acid construct that includes atleast a unique N-mer portion and a functional N-mer portion. Forexample, the unique N-mer portion can be used to tag—by means of itsunique sequence information-any target material labeled with thatconstruct. The functional N-mer portion may be used to attach theconstruct to a target (e.g., cells, proteins, nucleic acids, othermolecules, solid substrates, and other barcode constructs). Where, forexample, the target material includes nucleic acid, the functional N-mercan include a complementary nucleic acid (primer, hexamer, randomer,universal primer, etc.) to hybridize to the target. In some embodiments,the unique N-mer (“barcode sequence”) is attached to the functionalN-mer (e.g., “primer”) such that the barcode sequence is incorporatedinto a 5′ end of the primer. Alternatively, the barcode sequence may beincorporated into the 3′ end of the primer.

In some embodiments, more than one type of barcode-type label areincluded, for example, to be cross-combined. In one illustrativeembodiment, nucleic acid constructs of the invention are combined (e.g.,a set of constructs is cross combined one of each at a time with a setof other labels) with a labels of another type, such as magneticbarcoded beads from Applied BioCode.

The functional N-mer can operate as a primer sequence or sticky end tohybridize to nucleic acid. Functional N-mers can be designed to favorhybridization under certain conditions. For example, length or GCcontent can be varied to favor high-temperature (or stringent)conditions. Where a nucleic acid construct (barcode) will be made withsticky ends to append to other constructs, the melting temperature ofthe sticky ends can be tuned, for example, by tuning the length of thoseends. No particular length is required in general, so any given lengthcan be chosen based on intended melting temperature or other designconsiderations.

Attaching barcode sequences to nucleic acids is shown in U.S. Pub.2008/0081330 and PCT/US09/64001, the content of each of which isincorporated by reference herein in its entirety. Methods for designingsets of barcode sequences and other methods for attaching barcodesequences are shown in U.S. Pat. Nos. 6,138,077; 6,352,828; 5,636,400;6,172,214; 6,235,475; 7,393,665; 7,544,473; 5,846,719; 5,695,934;5,604,097; 6,150,516; RE39,793; 7,537,897; 6,172,218; and 5,863,722, thecontent of each of which is incorporated by reference herein in itsentirety.

Barcode sequences typically include a set of oligonucleotides rangingfrom about 4 to about 20 oligonucleotide bases (e.g., 8-10oligonucleotide bases), which uniquely encode a discrete library memberpreferably without containing significant homology to any sequence inthe targeted genome. The barcode sequence generally includes featuresuseful in sequencing reactions. For example the barcode sequences aredesigned to have minimal or no homopolymer regions, i.e., 2 or more ofthe same base in a row such as AA or CCC, within the barcode sequence.The barcode sequences are also designed so that they are at least oneedit distance away from the base addition order when performingbase-by-base sequencing, ensuring that the first and last base do notmatch the expected bases of the sequence.

Synthesis of oligonucleotides for use as constructs (e.g., barcodes orfunctional portions) can be by any means known in the art.Oligonucleotides can be synthesized on arrays, or in bulk, for example.

In certain embodiments, the barcode sequences are designed to becorrelated to a particular patient, allowing patient samples to bedistinguished. The barcode sequences incorporated into a plurality ofprimers (and subsequently into DNA or RNA targets) within a singledroplet may be the same, and vary from droplet to droplet.Alternatively, the barcode sequences incorporated into the plurality ofprimers (and subsequently into DNA or RNA target) within a singledroplet may be different. Designing barcodes is shown U.S. Pat. No.6,235,475, the contents of which are incorporated by reference herein intheir entirety. In certain embodiments, the barcode sequences range fromabout 2 nucleotides to about 25 nucleotides, e.g., about 5 nucleotidesto about 10 nucleotides. Since the barcode sequence is sequenced alongwith the template nucleic acid to which it is attached, theoligonucleotide length should be of minimal length so as to permit thelongest read from the template nucleic acid attached. Generally, thebarcode sequences are spaced from the template nucleic acid molecule byat least one base (minimizes homopolymeric combinations).

Methods of the invention include attaching the barcode sequences to afunctional N-mer such as a primer, then incorporating the barcode into atarget, or portion thereof using, for example, multiple displacementamplification. The labeled strands produced by MDA are able to befragmented or sheared to desired length, e.g. generally from 100 to 500bases or longer, using a variety of mechanical, chemical and/orenzymatic methods. DNA may be randomly sheared via sonication, e.g.Covaris method, brief exposure to a DNase, or using a mixture of one ormore restriction enzymes, or a transposase or nicking enzyme. RNA may befragmented by brief exposure to an RNase, heat plus magnesium, or byshearing. The RNA may be converted to cDNA before or afterfragmentation.

Barcode Droplet Libraries

In certain embodiments, the invention provides libraries of barcodes indroplets, as well as methods of making and using them. Making a barcodelibrary is shown in FIGS. 5A-FIG. 9B. A barcode droplet librarygenerally is a set of droplets containing barcodes (e.g., unique N-mers)for incorporation into a target molecule. Barcodes can be provided in anoligonucleotide containing sequence to function as an amplificationprimer with the result that a nucleic acid subsequently introduced intothe droplet will be amplified, and the copies that result will includethe barcode of that droplet. However, barcodes can also be provided thatare used to label proteins or other molecules of interest.

In various embodiments, there is a distinction between a droplet librarythat is used directly with samples (function N-mer is PCR primer, randomhexamer, etc), and a library that can be used either for continuedbuilding of higher complexity composite barcodes, or directly withsamples that have been prepared to contain appropriate sticky-ends(functional N-mer is a sticky end; the haplotyping with annealed samplesis one example of this case).

Regardless of the library type, the functional N-mer can be chosen basedon a type of target material. For example, for barcoding antibodies, oneset of antibodies could all have a sticky-end that binds one class ofbarcodes, and another antibody set would have a different sticky end,for example, to bind a capture tag. In another example, a set ofbarcoded PCR primers could include one forward/reverse pair that couldbind to one class of barcodes and a different ‘universal’forward/reverse pair that binds to a different class of barcodes (withthe compliment to the second for/rev pair).

Barcodes can be provided as oligonucleotides as discussed above. Incertain embodiments, a barcode is provided as part of a tripartiteconstruct (e.g., as shown in FIG. 7A-B) including a universal primingsite, a barcode, and a sequence specific region. The sequence specificregion can provide a PCR primer of known sequence, a random hexamer forMDA, or any other suitable nucleotide sequence that will bind to target.In other embodiments, the invention provides universal barcode libraries(e.g., droplets that each contain a plurality of universal primers orpriming sites all having a single unique barcode, but without asequence-specific region). A universal barcode generally includes aunique N-mer and a sticky end.

For creation of a library, a number of different barcodes will beobtained. For any given length, L, in nucleotides, the number N ofunique barcodes that can be made using standard nucleotides (A, T, C, G)is given by N=4^(L). It can be seen by simple calculation, for example,that if barcodes are to be five nucleotides long, then 1,024 uniquebarcodes are possible. Six, seven, and eight nucleotides in a barcodeallow for 4096, 16384, and 65536 unique barcodes, respectively. If eachbarcode includes 10 nucleotides, then more than one million uniquelibraries can be made. At 15 nucleotides, then N is greater than onebillion. Combining such barcodes using sticky ends (shown in FIGS. 5A-B)gives N′=N×N. In creating a barcode droplet library, a number ofdroplets are formed, each preferably containing copies of auniquely-barcoded construct.

For embodiments in which primer pairs are used, for example, wheretarget nucleic acid is to be amplified using PCR, one step of creating abarcode droplet library involves creating a forward library. In atripartite construct-based embodiment, each droplet in the forwardlibrary will contain a plurality of copies of uniquely-barcodedtripartite “forward” primers. That is, each tripartite construct in theforward library will comprise 5′-universal forward tail-barcode-forwardprimer-3′. While any number of droplets can be made in the forwardlibrary, in a preferred embodiment, the forward library contains setsthat include a number of droplets equal to or less than the number ofpossible unique barcode given the number of nucleotides in each barcode.Thus, if a six nucleotide barcode is to be used, sets of approximately4,000 droplets (or any arbitrarily-lower number) can be made.

A corresponding number of reverse tripartite constructs can be made(e.g., universal reverse tail-barcode-reverse primer). Then,microfluidic methods and devices as discussed herein can be used to addreverse constructs to each droplet containing forward constructs.Forward and reverse constructs can be put into droplets together in avariety of ways. For example, the forward and reverse constructs can beput into droplets a single well at a time. In some embodiments, flowingmicrofluidic systems are used. For example, a stream containing reverseconstructs can be merged with a stream containing the forward droplets.As each droplet passes the merge point, the reverse construct is added.

Forward and reverse constructs can be put together randomly, or they canbe put together in a serial fashion. In a serial approach, the firstreverse construct can be added to all droplets (e.g., about 4,000) of aset of forward droplets by flowing those droplets through the mergepoint. Then, the second reverse construct can be used, and the stepsrepeated. A second complete set of forward droplets can be streamed intothe second reverse construct, thereby creating 4,000 droplets, each ofwhich contains a unique forward primer and the second reverse primerconstruct. After this process is repeated 4,000 times, 4,000×4,000droplets will have been made, each containing uniquely-barcoded primerpairs (e.g., as tripartite constructs). Production of a large barcodelibrary by these means need not include tripartite constructs and canuse any constructs that include barcodes (e.g., primer pairs+barcodes;random hexamers+barcodes; universal primers+barcodes; etc.).

Where primer pairs are used, any number of primers or primer pairs canbe used. Where a large number of cells will be assayed for informationabout a single locus of interest, a single PCR primer pair may be usedin a large barcode droplet library. Where a barcode droplet library willbe used to assay a number X of loci on a plurality of genomes, X primerpairs will be used. Where MDA will be used to amplify one or more targetregions, a number of random hexamers will be used according tocalculations discussed elsewhere.

In certain embodiments, only one type of construct is provided perdroplet (i.e., forward only or reverse only, without a correspondingreverse). Thus, methods of the invention include preparation of barcodedroplet libraries in which each droplet contains a single barcodedconstruct without a corresponding partner-pair barcode.

In certain embodiments, primers for an initial round of amplificationare universal primers, for example, where the target to be amplifiedincludes universal priming sites.

As discussed elsewhere herein, droplets of the invention are stable whenstored. Thus a barcode droplet library can be prepared having anyarbitrarily large size and stored to be later used in any of the suitedassays described herein or known in the art.

In some embodiments, the invention provides methods involving a two-step“drop” PCR wherein multiple sets of primers are provided in a droplet.Either, both, or neither set of primers can include barcodes. Targetmaterial is added to the droplet. A first round of amplification isperformed, and then a condition is changed, and amplification isperformed again. For example, low-stringency conditions are created forthe first amplification, through manipulation of temperature or chemicalenvironment. Thus, even though other primers are present, an intendedfirst set of primers outcompetes or predominates in amplification. Bythese means, target nucleic acid can be amplified and barcoded inmultiple steps.

As discussed above, a barcode library generally includes constructshaving a functional N-mer and a unique N-mer. In some embodiments, afunctional N-mer is a sticky end.

The invention provides methods and materials to generate large, complex,or extensible barcode libraries, and applications for barcode libraries.

In order to facilitate generation of a sufficiently high number ofbarcoding oligonucleotide species for labeling a wide range ofmolecules, particles, or cells, one can generate a “Universal BarcodingDroplet Library” for combining with samples. This reagent can be used tobarcode DNA, RNA, proteins, chemicals, beads or other species present inthe sample if they contain complimentary binding moieties.

The concepts for generation and use of a droplet library for massivelyparallel molecular barcoding apply to all forms of binding agents thatcan have a readable identifying barcode appended. Expanded ‘plex’ forbarcode identifiers is provided via the use of barcodes in droplets,such that one barcode can be linked to other barcodes via one or morelibrary combinations, resulting in multiplicatively larger sets ofunique barcodes.

In certain embodiments, antibodies or oligonucleotides are used asfunctional N-mers for binding to sample molecules with (optionallyreleasable) unique N-mers as barcodes. Both the types and numbers ofeach type of barcodes are determined by a digitally quantified readout,and thus correlated with the presence and concentration of variousbiomarker species in a sample.

Two basic types of universal barcoding droplet libraries are describedas examples of the general concept for providing a means to appendunique barcodes to target material for identification or quantification,but the concept is not limited to these examples and at least oneexample will be given where the two described library types are usedtogether.

In the first set of examples, a universal binding barcode dropletlibrary is described for use in a ‘bind and ligate’ approach (see FIG.6). This library type consists of droplets containing oligonucleotidestrands that encode barcodes and contain ligation competent ends,enabling the modular linking of barcodes by specific hybridization (alsoreferred to as ‘annealing’ or ‘binding’) in droplets followed byligation into a covalently bonded strand (or duplex) of bases. TheUniversal Binding Barcode Droplet Library can be used directly withsamples that contain pre-bound barcoded binding moieties, as a ‘primary’library that is combined with binding moieties targeting specific samplemolecules, or can be used in the construction of ‘secondary’ or higherorder binding barcode libraries through the successive combination ofdroplet libraries. The end use of such libraries can include assembly ofthe barcoded specific binding agents into a release-able and readablesingle molecule for use in digital quantification of bound targets for avariety of applications.

In the second set of examples, a universal priming barcode dropletlibrary is described for use in a ‘bind and prime’ approach. FIGS. 7A-Bshow one example of a universal barcode droplet library with targetingprimers (e.g., to “bind and prime”). This library type consists ofdroplets containing barcoded primers for PCR (or other polymerase)priming, such that after combination with a sample droplet containing atleast one target sequence from the same single DNA or RNA molecule, ormultiple molecules co-localized in a single droplet, a digitallyreadable oligonucleotide barcode is attached to the target molecule'ssequence. Since all polymerase generated molecules in the same dropletwill have the same barcode, the co-localization information is retainedafter release from the droplet, and any sequencer can be used to bothdetermine the sequence and count the number of templates traceable toeach original droplet.

Both library types enable molecular barcoding in droplets, providing alarge excess of unique identifying barcodes compared to the number ofsample droplets, or compared to the number of sample objects ormolecules contained in the droplets, thus allowing digitalquantification of many targets of interest on various reading platforms.Significantly, the two types are not exclusive of each other. Forexample, FIG. 9A-B shows ligating sticky-ended universal barcodes tobarcoded PCR primers.

Sticky End Libraries

FIGS. 5A-B show the overall scheme for construction of a universalbinding barcode droplet library. Pairs of overhanging complimentaryoligonucleotide barcodes are chemically synthesized (using standardcommercial manufacturing methods) such that the complementary barcodingsequences are flanked by ‘sticky-ends’ for subsequent annealing andligation to the target species or other barcodes, or for polymerase orother enzymatic priming. The oligonucleotides may include 5-prime or3-prime phosphorylation, or combinations of these or othermodifications. Methods to make oligonucleotides resistant to nucleaseactivity may be used, including the use of 2′O-Methyl RNA bases and/orphosphorothioated bonds to form the entire backbone of the oligo or tocap the ends of the sequence. PNA, LNA, or other modified nucleotidestructures can also be used. A sticky-end may be any length andsequence, with preferred embodiments containing base pairs includingrestriction endonuclease cleavage sites, or priming sites for sequencingor digital PCR, or array hybridization, and any number of sticky-endswith different sequences can be utilized. Sticky-end sequences may beused as barcode identifiers as part of composite barcodes.

Two example barcoded oligonucleotide pairs are shown in FIGS. 5A-B (1aand 2a, flanked by sticky-end Type1 and sticky-end Type2). To constructa droplet library each discrete complementary oligonucleotide pair canbe placed together into a standard microtiter-plate well and formed intodroplets, which can be subsequently mixed with other oligonucleotidepair-containing droplets to make a ‘primary barcode droplet library’.Forming droplets for a library is discussed in U.S. Pub. 2010/0022414.The number of pair types (N members) is not limited.

These storable stable droplets can either be used directly as anN-member barcoding library, or combined with another barcodingoligonucleotide set (M-members) to form a ‘tandem’ barcoded library withN×M=NM-plex. A 4000 N-member library combined with a 4000 M-memberlibrary will generate a 16 million-plex barcode library.

Combination of the N-member primary barcode library with the M secondarybarcodes can be done in series (with each member of the M-barcodecombined as an aqueous liquid one at a time with the N-member primarybarcode library, using various methods including lambda orpico-injection modes and co-flow) or by combining the N-member andM-member library droplets in parallel (primary library combined withsecondary library).

Heterogeneous mixtures of barcodes (e.g. barcodes synthesized usingdegenerate bases) can be converted into a unique set of droplet barcodesby addition of a unique sticky-end. Manipulation of droplets isdescribed in U.S. Pat. No. 7,718,578 and U.S. Pub. 2011/0000560.

By combining complimentary sticky-ends from two barcode sets, the fouroligonucleotide types present in the final combined droplet willspecifically hybridize to create a sticky-ended tandem barcode (e.g.,droplet 1 or 2 in FIG. 5A-B). This can then be ligated together. Asimilar specific hybridization will occur for additional numbers ofbarcodes containing complimentary sticky-ends. This is illustrated inFIG. 6A-B, with ‘single sticky-ended’barcoded oligonucleotide pairsshown on the left, where one end is capped such that there is nooverhang, and ‘double sticky-ended’ barcode oligonucleotides shown inthe middle panel (either different or similar sticky-ends can be used,with different ends precluding promiscuous concatamer formation).Additional modifications of the sticky-ends can also be included (e.g.biotin or desthiobiotin, shown on the bottom left of the figure).

After annealing the sticky ends together, adjacent strands can beligated together.

The panel on the right of FIGS. 6A-B shows the initial binding barcodedroplet library (only one droplet and one molecule of each type shown,with a barcode identifier 1a) on the top, a tandem barcoded dropletlibrary formed by combination of a primary barcode and a secondarybarcode in the middle (e.g. barcode identifier 1a: 1b), and a triplebarcoded library at the bottom (formed by combining a secondary barcodedlibrary with a third barcode, resulting in barcode identifier 1a:1b:1c).

This modular construction is not limited to the combinations shown, withany composite sticky-ended barcode library able to be combined withadditional barcodes in subsequent rounds of droplet combination. Even alow number of combinations can result in a very high level ofbarcode-plex.

For example, a 16 million-plex tandem barcode library (made from 4000N×4000 M barcoded oligos) can be combined with another sticky-ended setof 4000 Z barcoded oligos to form a 64 billion-plex barcode library (16million NM members×4000 Z-members=64 billion). As shown in FIGS. 6A-B,the oligonucleotides can be designed such that the resulting annealedoligo set can have a single or double sticky-ends (with different orsimilar ends).

A barcode library can also be made to include a sticky-end adapterspecific for a sequencing platform. In certain embodiments, a constructis made that includes a sequencing platform N-mer and a sticky-endN-mer. A library of these constructs can be made. Separately, auniversal barcode library as discussed above can be made. The, theuniversal barcode library can be combined with the sequencing platformadapter library by means of the sticky ends in view of a particularapplication. Thus products of any analysis discussed herein can beadapted to go directly into the workflow of any given sequencingplatform (e.g. sticky-ended Illumina adaptors to anneal/ligate ontoeither the primer library or the output from a targeted sequencing run,so that it could be hybridized directly onto their flow cell. Adifferent sticky-end adaptor set could be used for 454, etc.). Thisapproach can minimize PCR bias.

A universal PCR primer barcode library can also be prepared with anunlimited amount of plex by creating sticky-ended forward and reverseprimers that can be further combined with additional numbers ofsticky-ended barcodes to generate combinatorial barcodes, as shown atthe top of FIGS. 9A-B. The forward and reverse universal primers areconstructed in an identical fashion as described above and in FIGS. 7A-B(primary barcoded primers) and then annealed to a sticky-ended barcodeoligonucleotide pair (either single or double sticky-ended as shown inFIGS. 5A-B) and subsequently ligated, to make a contiguous forward(and/or reverse) primer annealed to the complimentary oligo that wasused to anneal to the primary barcoded primer. The top right side ofFIGS. 9A-B shows the ligated product after addition of both forward andreverse single sticky-ends to create a ‘secondary’ barcoded priming set.The bottom of FIGS. 9A-B shows a single droplet after combination of oneprepared template-containing sample droplet with one universal PCRprimer barcode library droplet. The annealed PCR primers can beamplified by using polymerase and dNTPs, and all of the amplicons fromthis droplet will be barcoded 1b:1a:1a:1b (with 1b:1a 5-prime to thetarget loci sequence, followed by 1a:1b, as read 5-prime to 3-prime).

Haplotype Phasing

The invention provides systems and methods for haplotype phasing andgenotyping. A nucleic acid can be isolated from a sample and haplotypedthrough the examination of a number of loci. For example, the allelicform present for a number of suspect SNPs of interest can be determinedalong a single chromosome. By including barcodes in multiplexed tiledPCR reactions within droplets, this aspect of the invention enables‘haplotype phase’ assignments to be made using existing sequencingplatforms.

Several aspects of the invention are combined to enable assignment ofsequencing information (e.g., a series of SNPs) to target DNA stretches.In certain embodiments, haplotype phasing involves preparing a barcodedroplet library in which each droplet contains the set of primers toamplify the loci of interest. Preferably, each primer is part of atripartite construct that also includes a universal priming site (forsubsequent amplification, capture, or sequencing) and a barcode. In eachdroplet, every tripartite construct will preferably contain the samebarcode. Multiplexed PCR primers that will not cross-hybridize with eachother and which will uniquely amplify the target DNA locus can be used.

The overall workflow example is shown in FIG. 10A-B. Optionally, thetarget locus can be pre-amplified using a single pair of PCR primersthat flanks the entire locus, before appropriate loading of the sampleinto droplets for amplification and barcoding (not depicted in theworkflow in FIG. 10A-B).

Preferably, each tripartite construct includes a universal tail portionimmediately 5′ to a barcode sequence followed by the sequence-specifictargeting primer bases. A primer droplet library ‘member’ includes adroplet that contains all of the targeted primers sufficient forcovering the target bases, each with the same barcode that will enablepost-sequencing correlation to the target strand. The number of librarymembers is determined by the ratio of barcode number to the number oftarget alleles to be analyzed. By way of example, without limitation,FIG. 10A-B shows 100 cells as input, with 4000 barcodes giving a 1/10chance of duplicate barcodes for any allele. In this example, the DNAfrom 100 cells provides 400 target alleles, which are loaded (togetherwith polymerase, buffer, and nucleotides) into one million droplets andcombined with the barcoding primer library to generate a PCR-competentdroplet emulsion. As an example for a 3 kb target region, 13 tiledprimer pairs can be used to cover the target bases. Fewer primer pairscan be used if only subsets of the target bases are to be phased.

Droplets from the droplet library are merged with target nucleic acid.In the example pictured in FIG. 10A-B, genomic DNA has been sheared to alength of about 20 kilobases. Fragments of the target are introducedinto droplets such that on average each droplet will generally containno more than one molecule of target nucleic acid.

After this merging step, each droplet contains at least: a pair oftripartite constructs for each locus of interest; a single nucleic acidmolecule; and PCR reagents (e.g., Taq, dNTPs). Each droplet is thenthermocycled, producing numerous copies of each locus of interest inwhich each of those copies has a unique pair of barcodes at each end.

Droplet libraries are generally discussed herein as having pairs ofbarcodes and in some embodiments those barcodes may be necessarily thesame or different, while in other embodiments, it may not matter whetherthe forward and reverse barcodes are the same or different. For example,if barcodes have six nucleotides, and millions of targets are to beanalyzed, then using different forward and reverse barcodes will createample unique barcode pairs. Here, an embodiment of haplotype phasing ispresented in which the forward and reverse barcodes need not be thesame. In some embodiments, those bar codes may be the same.

After thermocycling, the amplified products are released into a singlebulk aqueous phase (e.g., using a droplet de-stabilizing reagent), and asubsequent amplification, sequencing, or capture is performed using theuniversal primer tail as well as any sequencing platform-specificadaptor (and additional barcodes) needed before sequencing. Examples ofthe PCR inputs and outputs are shown in FIG. 11A-C. Using a yieldthreshold of ˜150 sequencing reads as being more than sufficient forhigh confidence SNP calling, the total number of PCR cycles (droplet PCRplus bulk PCR) can be limited to 10 cycles (sufficient to generate 150copies).

The amplified products are then sequenced according to any method knownin the art including those discussed herein. Even though, in certainembodiments, the amplified products are provided for sequencing in abulk aqueous phase, sequencing results can be haplotyped based on thepresence of the barcode in the sequence reads. Particularly inembodiments in which the barcode is immediately 3′ to a sequencingprimer binding site, every sequence read will have barcode information.

In certain embodiments, genetic material is haplotyped withoutamplification as shown in FIG. 15A-FIG. 17B. FIG. 8A-B shows the overallscheme for construction of a universal priming barcode droplet libraryfor use in a ‘Bind and Primer Extend’ application (addition of stickyend component on right of FIG. 8A-B is optional). Primers as shown inFIG. 8A-B can be used as shown in FIGS. 15A-1B to provide haplotypeinformation without amplification. In this example, a the initial‘primary’ droplet library contains single universal priming barcodes forprimer extension, constructed using similar methods as that describedfor construction of a universal binding barcode droplet library.Contiguous oligonucleotides are chemically synthesized with each of thesequence components including a universal forward tail (consisting ofbases to be annealed to an analogous sticky-end), a sequence of basesused as a barcoding component, and a priming sequence that will annealto the universal 5-prime end of the target sequence to be counted (thecomplimentary target is designated as ‘for’ in, for example, FIG.15A-FIG. 17B).

FIG. 15A-B shows up-front processing for haplotyping a single moleculeof DNA using a universal priming barcode library. A forward primer isused with a corresponding oligonucleotide (here labeled 3′ Tailedprimer). Note that the corresponding 3′ oligonucleotide need notfunction as a primer. For convenience, they will be referred to asprimers. To prepare for target molecule haplotyping, a set of specifictargeting primers are prepared and annealed to the target DNA, with a 5′oligonucleotide comprising oligonucleotides complimentary to the 5′ endof the target locus (3 loci are shown in FIG. 15A-B) and a universal‘for’ sequence that will be used to anneal to the barcode library, and a3-prime oligo comprising oligos complimentary to the 3-prime end of thetarget locus followed by a universal 3-tailed end. The target moleculeto be haplotyped can be targeted by any number of primer pairs andtargeted loci can be any distance from other loci (the shear length ofthe genome will limit the maximum distance that can be haplotyped).

Each of the uniquely barcoded primers can be reformatted from a wellplate into a droplet format, either for direct use with samples or to beused in optional subsequent rounds of droplet combination to addcomplimentary sticky-ended barcoding sequences (right side of FIG. 8A-B,also shown in right side of FIG. 9A-B). Combination of the primary‘forward barcode library’ with sticky-ended barcodes (either serial orlibrary combination) provides another barcode encoding level.Alternatively, the primary droplet library can be generated from pairsof primers initially placed together in wells and reformatted intodroplets, with a sticky-ended barcode oligo together with variousforward barcoded primers. Additional rounds of droplet combination witheither single or multiple sets of sticky-ended barcode members can beused to get to any required level of barcode-plex needed for the assay.

A universal priming barcode library having sufficient plex to uniquelybarcode the sample targets is used by combination and annealing totarget molecules that have been either prepared in bulk or in droplets(bulk prep is shown in FIG. 15A-B).

Hybridization of all of the targeting primer pairs with the targetsample is followed by removal of unbound primers (e.g. purification bysize or via use of biotinylated primers or targets), elongation of theforward hybridized primer for each locus using a polymerase (e.g.Klenow) that lacks exonuclease activity such that the elongation stopswhen the polymerase encounters the 3-prime primer, and subsequentligation using ligase and ATP or photo-ligation or other ligation means.The resulting bulk sample preparation contains the target singlestranded molecules with each targeted loci annealed to a contiguousoligonucleotide strand that contains the newly synthesized compliment tothe target loci bracketed by the added primer pairs.

This output of the process shown in FIG. 15A-B is the target which thenucleic acid constructs (primers) shown in FIG. 16A-B bind to.

The bulk annealed sample prep is loaded into droplets, along withpolymerase and dNTPs, such that a single contiguous molecule with itsset of targets to be haplotyped is contained in a droplet. The UniversalBarcode Library is combined with the prepared sample, either in adroplet to droplet combination mode, or in a droplet to aqueous streamcombination mode (shown in FIG. 16A-B). FIG. 16A-B shows introducingprimer pairs, each bearing a barcode.

As shown in FIG. 16A-B, a pair of primers for each locus of interest isincubated with a strand of target DNA and allowed to hybridize. Anyunbound (un-hybridized) material is removed. The two forward ‘for’primers can be elongated either inside droplets that include thepolymerase and dNTPs, or following release of the primers annealed tothe template (the workflow in FIG. 17A-B shows release before elongationoutside of droplets). Polymerase synthesizes a complementary strand fromthe 5′ forward primer to the 3′ “reverse primer”. Ligase then ligatesthe complementary strand to the 3′ reverse primer. As a result, acomplementary strand has been synthesized representing each locus ofinterest.

The droplets can be lysed (or “burst”, discussed elsewhere herein), andthe contiguous strands (e.g., barcoded amplicons) released into bulkphase as shown in FIG. 17A-B.

The resulting contiguous strands containing barcodes are used forsequencing (or sequence determination using microarrays), and thesequences that contain identical barcodes have sequences that derivefrom the same sample strand (i.e. the same haplotype). This exampleshows a method for haplotype determination without any amplification(i.e. only elongation and ligation), and has the advantage ofelimination of potential amplification bias. In addition, if a locus hascopy number variation within the same sample target molecule, this canbe seen as variation in the number of sequenced reads that have the samebarcode and sequence.

In contrast to the “no-amplification” haplotype methods, the inventionalso provides methods for single-molecule haplotyping that includeamplification. In certain embodiments, amplicons can provide targetmaterial according to a “bind and primer PCR” approach.

FIGS. 7A-B show the overall scheme for construction of a UniversalPriming Barcode Droplet Library for use in a ‘PCR’ version of thehaplotyping application, which provides haplotype information withamplification. In this example, the initial droplet library containssingle ‘forward’ universal priming barcodes that will be subsequentlypaired with a ‘reverse’ barcoded primer to create a Universal PCR PrimerBarcoded Droplet Library. Contiguous oligonucleotides are chemicallysynthesized with each of the sequence components including a universalforward tail (consisting of bases to be used as priming sites or forligation to adaptors used for sequencing), a sequence of bases used as abarcoding component, and a forward priming sequence that will anneal tothe universal 5-prime end of the target sequence to be counted (thecomplimentary target is designated as ‘for’ in FIG. 12A-FIG. 14B). Eachof the uniquely barcoded primers is reformatted from a well plate into adroplet format. Combination of the forward primer barcoded dropletlibrary with similarly constructed reverse primer barcodes (eitherserial or library combination, with the serial mode shown in FIG. 7A-B)generates the universal PCR primer barcoded droplet library.Alternatively, the a PCR primer droplet library can be generated frompairs of forward and reverse primers initially placed together in wellsand reformatted into droplets, however this type of library will havethe same number of unique forward/reverse barcode combinations as thenumber of paired primers wells.

A universal priming barcode library having sufficient plex to uniquelybarcode the sample targets is used by combining with samples andannealing to complimentary target molecules present in the sample. Anexample sample preparation scheme is shown in FIG. 12A-B. To prepare fortarget molecule haplotyping, a set of specific targeting oligos areprepared and annealed in bulk phase to the target DNA, with a 5-primeoligonucleotide comprising oligonucleotides complimentary to the 5-primeend of the target locus (3 Loci present on the same contiguous targetmolecule are shown being targeted for haplotyping) and a universal ‘for’sequence that will be used to anneal to the Universal Priming BarcodeLibrary, and a 3-prime oligo comprising oligos complimentary to the3-prime end of the target locus followed by a universal 3-tailed end.The target molecule to be haplotyped can be targeted by any number ofprimer pairs, and the targeted loci can be any distance from each other(the shear length of the genome will limit the maximum distance that canbe haplotyped). Hybridization of all of the targeting primer pairs withthe target sample is followed by removal of unbound primers (e.g.purification by size or via use of biotinylated primers or targets),elongation of the forward hybridized primer for each locus using apolymerase (e.g. Klenow) that lacks exonuclease activity such that theelongation stops when the polymerase encounters the 3-prime primer, andsubsequent ligation using ligase and ATP or photo-ligation or otherligation means (bottom of FIG. 12A-B). The resulting bulk samplepreparation contains the target single stranded molecules with eachtargeted loci annealed to a contiguous oligonucleotide strand thatcontains the newly synthesized compliment to the target loci bracketedby the added primer pairs.

Use of the universal PCR barcode primer library with prepared sample isshown in FIG. 13A-B. The bulk annealed sample prep is loaded intodroplets, along with polymerase and dNTPs, such that a single contiguousmolecule with its set of targets to be haplotyped is contained in adroplet. The universal barcode library is combined with the preparedsample, either using a droplet to droplet combination mode, or in adroplet to aqueous stream combination mode (shown in FIG. 13A-B).

The two forward ‘for’ complimentary base pairs are allowed to hybridize(two droplets from a combined library and sample are shown in FIG.13A-B, and elongated either inside droplets that include polymerase anddNTPs, or following release of the primers annealed to the template (theworkflow in FIG. 14A-B shows release before elongation outside ofdroplets), followed by ligation of the 3-prime primer to the elongatedstrand. The resulting contiguous strands shown on the right side of FIG.14A-B containing barcodes are used for sequencing (or sequencedetermination using microarrays), and the sequences that containidentical barcodes have target loci sequences that derive from the samesample strand (i.e. the same haplotype phase).

Single Cell Genomics

Genotyping of single cells can be performed in a fashion similar tohaplotyping discussed above. FIG. 18A-B depicts the steps associatedwith isolation, encapsulation, molecular labeling, sorting and analysisof single cell genomes using fluidic droplets (including optionalupfront sorting, and cell lysis within droplets using a detergent andheat).

Preferably a first library of droplets is formed, each dropletcontaining genetic or proteomic material (e.g., from a single cell, or aportion thereof). In certain embodiments, single cell droplets arecreated and then the cells are lysed within the droplets.

The droplets can be merged with droplets from a barcode droplet library(e.g., containing nucleic acid constructs in which the functional N-mersare primer oligonucleotides to hybridize to the genome of the singlecell). As a result, each droplet will contain at least: the entiregenome from a single cell; nucleic acid constructs for hybridizing tothe genome of interest with a barcode; and analysis reagents (e.g.,polymerase and nucleotides).

A second library of droplets is formed, each containing a plurality ofN-mers, each N-mer containing an associated label. In a preferredembodiment, each droplet in the second library of droplets contains thesame label within the given droplet, and the labels preferably vary fromdroplet to droplet. Each droplet in the second library further containsreagents for the replication of the genetic material in the firstdroplet and subsequent incorporation of the tag. The replication can beDNA from DNA or DNA from RNA (cDNA). There can be a single replicationof the genetic material, there can be a linear amplification of thegenetic material or an exponential amplification of the genetic materialsuch as PCR or multi-strand displacement amplification. The reagents forconducting the replication can include such things as polymerase,reverse transcriptase, nucleotides, buffers, etc.).

Alternatively, the second droplet could contain beads which are designedto capture the target nucleic acid. Capture sequences with a tag can beattached to bead and used to capture the genetic material from the firstdroplet introduced after merging with the second droplet. The capturesequence with the tag can be synthesized directly onto the beads or beattached by such means as biotinylated sequences and streptavidin beads.The use of streptavidin beads and biotinylated sequences has theadvantage of allowing a generic bead to be used with new libraries ofbiotinylated capture sequences that can be assembled on demand. However,any method known in the art for attaching nucleic acid sequences tobeads can be utilized.

Alternatively, the second droplet could contain capture sequences thathave an attached molecule that is capable of being captured on a solidsurface. Biotin would be such a molecule that could be captured bystreptavidin attached to a solid surface. Other methods known in the artsuch as antibody/antigen or aptamers could also be utilized.

The target regions of the genome can be amplified according to theworkflow shown in FIG. 18A-B. Amplification of the target region of thegenome can be accomplished by any method known in the art. For example,amplification and barcoding can involve barcoded primers, as shown inFIG. 19A-B. FIG. 20A-B shows single cell genomics using primers for afirst round of amplification followed by subsequent merging with auniversal barcode library. Primers as shown in FIG. 19A-B or in FIG.20A-B generally can amplify target material by a PCR reaction.

The new library of droplets then undergoes amplification (see “lst RoundPCR” in FIG. 19A-B) to incorporate the labels into the genetic target(i.e., DNA or RNA).

Target material can also be amplified using multiple displacementamplification (MDA) or any other method known in the art. In certainembodiments (e.g., certain MDA embodiments), a random hexamer (orsimilar oligo) is used in conjunction with a barcode. For example, FIG.21 shows generating a barcoded random hexamer library and using therandom hexamer library with phi29. FIG. 22 shows a barcode/primerlibrary used with transposase.

In the description that follows, amplification is described in terms ofthe use of MDA for incorporating the labeled primers into or onto thetarget material, but it is understood that this choice of description isnot limiting for the invention, and that similar methods (e.g., using atransposase) are compatible with all other methods of the reaction.Where the target is genomic DNA, the label can be incorporated into theDNA using, for example, multiple displacement amplification with randomhexamer primers having a label incorporated at the 5′end.

Multiple displacement amplification (MDA) is a non-PCR based DNAamplification technique. This method can rapidly amplify minute amountof DNA samples to reasonable quantity for genomic analysis. The reactionstarts by annealing random hexamer primers to the template and DNAsynthesis is carried out by high fidelity enzyme, preferentially i29 ata constant temperature. Compared with the conventional PCR amplificationtechniques, MDA generates larger sized products with lower errorfrequency. MDA reaction starts with the annealing of random hexamerprimers to the DNA template and then continues with the chain elongationi29. Increasing number of primer annealing events happens along theamplification reaction. The amplification reaction initiates whenmultiple primer hexamers anneal to the template. When DNA synthesisproceeds to the next starting site, the polymerase displaces the newlyproduced DNA strand and continues its strand elongation. The stranddisplacement generates newly synthesized single stranded DNA templatefor more primers to anneal. Further primer annealing and stranddisplacement on the newly synthesized template results in ahyper-branched DNA network. The sequence debranching duringamplification results in high yield of the products. To separate the DNAbranching network, S 1 nucleases is used to cleave the fragments atdisplacement sites. The nicks on the resulting DNA fragments arerepaired by DNA polymerase I. The generated DNA fragments can bedirectly used for analysis or be ligated to generate genomic librariesfor further sequencing analysis. Using MDA, the DNA would simultaneouslybe amplified and have a barcode incorporated into the sequence.

Alternatively, if the second droplet contains beads to which areattached tagged sequences designed to capture the genetic material, themerged droplets are incubated in a manner that releases the geneticmaterial from a cell if present and allows the hybridization of thegenetic material to the capture sequences attached to the bead. Reagentssuch as proteases, alkaline reagents, detergents or other methods knownin the art can be used to release the genetic material from the cells ifpresent. After capture the emulsion is broken and the beads used topurify the genetic material away from other components of the reactionthat may inhibit subsequent steps such as cell debris, proteases anddetergents. The beads would capture the genetic material using attachedsequences that were random N-mers of length N designed to capture allsequences or variations of N-mers designed to capture only portions ofthe genetic material present. The tag is then incorporated byreplication of the captured nucleic acid using the capture sequence withthe tag as a primer. The replication can either generate DNA from a DNAstrand or DNA from an RNA strand (cDNA synthesis). This material caneither be processed directly or amplified further using methods known inthe art such as PCR or multi-strand displacement amplification.

The capture sequences can be synthesized directly onto the beads or beattached by such means as utilizing biotinylated sequences andstreptavidin beads. The use of streptavidin beads and biotinylatedsequences has the advantage of allowing a generic bead to be used withnew libraries of biotinylated capture sequences that can be assembled ondemand. However, any method known in the art for attaching nucleic acidsequences to beads can be utilized.

Alternatively, if the second droplet contains capture sequences with thetag and an attached binding molecule the merged droplets are incubatedin a manner that releases the genetic material from a cell if presentand allows the hybridization of the genetic material to the capturesequences with the binding molecule. Reagents such as proteases,alkaline reagents, detergents or other methods known in the art can beused to release the genetic material from the cells if present. Theemulsion is then broken to release the hybridized capture sequence andgenetic material. The released genetic material hybridized to thecapture sequence is then captured on a solid support allowing theremoval of elements such as cell debris, proteases and detergents thatmay inhibit subsequent steps. The tag is then incorporated byreplication of the captured nucleic acid using the capture sequence withthe tag as a primer. The replication can generate DNA from a DNA strandor DNA from an RNA strand (cDNA synthesis). This material can either beprocessed directly or amplified further using methods known in the artsuch as PCR or multi-strand displacement amplification.

Where the target is RNA, the label can be incorporated in the RNA usingMDA with random hexamers having barcodes attached at the 5′ ends.Alternatively, poly dT primers having barcodes attached at the 5′ endscan be used. A promoter region, such as T7 or SP6 RNA polymerasepromoter could also be incorporated at the same time, which could beused to amplify the RNA and incorporate a barcode.

After the target is labeled, the amplified genetic material can beanalyzed. For example, the amplified material can be released fromencapsulation (e.g., “Release from Droplet” in FIG. 19A-B) or from thebead or solid support and prepared for direct sequencing using, forexample, sequencing library preparation protocols well known to thoseskilled in the art. For example, the amplified genetic material can besheared/fragmented using methods well known to those of ordinary skillin the art, and adaptors can be ligated onto the ends of the fragmentsto be utilized, for example in direct sequencing, or in an enrichmentprocess.

While the entire encapsulated target can be tiled with a labeled primerusing the methods described herein, it should be noted that a majorityof the fragments input into the sequencing reaction may not contain alabel due to the shearing/fragmentation preparation step required forshort read sequencing technology. In other words, each of the MDAamplified labeled strands are sheared/fragmented such that one or morefragments from the same labeled strand become disassociated from thelabel incorporated into that strand (see FIG. 3A). This issue can beresolved in a number of ways. For example, once encapsulated the targetcan be fragmented prior to the amplification step to incorporate thelabels, thus ensuring that each fragment is labeled when it is inputinto the sequencing reaction. Alternatively, the MDA amplified labeledstrands can be enriched in a subsequent PCR reaction prior tosequencing.

In an exemplary embodiment, enrichment of the labeled strands can beaccomplished by incorporating a universal sequence into a 5′ end of eachof the plurality of labeled primers such that each of the primers has asequence as follows: 3′-(N-mer)-label-(universal priming sequence)-5′(see FIG. 3B). Once the labels are incorporated into the encapsulatedgenetic material (using, e.g., multiple displacement amplification, asdescribed above), the amplified material can be released fromencapsulation using one or more methods described in further detailbelow. A universal primer can then be ligated onto the 3′ ends of theamplified mix and input into a standard PCR reaction (FIG. 3B). Onlythose strands having the incorporated label with the universal PCRsequence will be amplified, and thereby enriched.

Alternatively, sequence-specific enrichment can be achieved using asimilar method to enrich for fragments that have barcodes and fortargeted regions of interest. For example, a plurality of labeledprimers each having a universal PCR sequence incorporated into a 5′ end(i.e., 3′-(N-mer)-label-(universal PCR sequence)-5′) is introduced intoa droplet containing the target. The labels are incorporated into theencapsulated genetic material using, e.g., amplification. The dropletcontaining the amplified mixture is then re-merged with another dropletcontaining a universal primer and a primer specific for a targetedsequence of interest, as well as reagents sufficient for conducting aPCR reaction. This merged droplet is then exposed to conditionssufficient for conducting a PCR reaction, and only those fragmentshaving barcodes and the targeted regions of interest will be amplified.

The labels incorporated within each droplet enables a plurality ofsequences from multiple different genomes to be simultaneouslyamplified, pooled for sequencing, then mapped back to their originalgenome/transcriptome. Like paired read/paired end sequencing, themethods of the invention provide a researcher/clinician/physician withthe ability to link two strands that are physically separated within asingle genome/transcriptome to the same genome/transcriptome by tilingthe same label along an entire genome/transcriptome. The skilled artisanwill readily recognize that reads from high repeat regions and/orregions of high homology can be easily mapped, haplotype information canbe obtained, and rearragements or deletions can be identified using themethods described herein. Additionally, mutations in subpopulations ofcells in a sample, and metagenomic loss of identity can be traced usingthe methods of the invention.

Barcoding Transcriptomes

The invention provides methods for analyzing transcriptomes that includelabeling transcriptomes with nucleic acid constructs comprising uniqueN-mers and functional N-mers. FIG. 23A-B is a schematic depictingvarious exemplary barcode schemes for the generation of a barcoded mRNAprimer droplet library. As shown in FIG. 23A-B, a construct can includea biotinylated universal primer, a barcode, and a poly-T region. Theseconstructs can be used to generate mRNA barcoding primer dropletlibraries wherein each droplet contains one or more copies of a uniqueconstruct. As shown in FIG. 23A-B, a variety of primer types can be usedto copy mRNA. Primer type variations include poly-T oligonucleotides,sequence specific primers, and random hexamers.

FIG. 23A-B shows a flow chart for barcoding mRNA. The mRNA is hybridizedto a barcoded primer, released from fluid compartments, purified onstreptavidin beads, and copied with reverse transcriptase. The resultingcDNA can be analyzed by sequencing.

FIG. 24 is a flowchart depicting the steps associated with isolation,encapsulation, molecular labeling, sorting and analysis of single celltranscriptomes using fluidic droplets (including optional upfrontsorting, and cell lysis within droplets using a temperature-inducibleprotease). As shown in FIG. 24, a sorting mechanism can be used to sortthe mRNA-containing droplets.

FIG. 25A-B shows a sorted cell workflow for barcoding transcriptomesfrom single cells using a barcode library in a detergent lysis buffer.FIG. 25A-B shows a workflow for barcoding transcriptomes from singlecells. FIG. 26A-B is an alternative flowchart depicting the stepsassociated with isolation, encapsulation, molecular labeling, sortingand analysis of single cell transcriptomes using fluidic droplets(capture beads are included in the droplet library).

With these described modifications for target material being mRNA, theabove described approaches to haplotyping and genotyping can be appliedto transcriptomes.

Biomarker Counting

In certain embodiments, a universal barcode droplet library (e.g., of abind-and-ligate type) is used to count biomarkers associated with asingle cell.

A barcode library can be used in combination with binding molecules thatare also barcoded to provide target identification, linking informationabout the type and number of target molecules present in a sample withtheir co-incident presence in the same droplet. FIG. 27A-C firstillustrates the construction of sticky-ended barcoded binders, and thenshows how they can be used for generating cell population averages ofthe identified biomarkers, before FIGS. 28-31 illustrates how the samesticky-ended barcoded binder constructions work using dropletizedversions of both the samples and the barcoded reagents.

The barcoded binding reagents are constructed by linking in a reversiblemanner a sticky-end oligonucleotide onto the binding reagent (e.g.antibody binding reagents are shown in FIGS. 27A-31D). Two examples oflinking motifs tying the barcode to the binder are shown in FIG. 32A-C,one using a photo-activatable linking base 5-prime to a photo-cleavablebase and the other using a restriction enzyme cleavage site 5-prime tothe barcode that identifies the binding species. By linking anoligonucleotide that contains a cleavable linker, an optional adaptorsequence (to enable interfacing with the downstream sequencing method),a barcode identifying the binding species, and a sticky-end forcombining with the complimentary sticky-ended barcode library to aparticular binding species, a ‘sticky-ended barcoded binder’ is created(e.g. barcoded binder#1 in FIG. 27A-C). A variety of commercial kits andreagents are available for linking the oligonucleotide to the binder(e.g. chemical cross-linking agents for linking to a protein binder,hybridization and ligation or synthesis of the entire sequence forlinking with an oligonucleotide binder). A second similar set of motifslinking a second binding species to a second identifying barcode withthe same universal sticky-end is constructed as ‘barcoded binder#2’.Additional barcoded binders are constructed such that a set of nbarcoded binders is available for use either to determine bulk averagequantification of biomarkers (FIG. 27A-C), or for droplet-basedquantification of single species (e.g. single cells, single capturereagents, etc.).

FIG. 27A-C shows the workflow for determination of biomarker averagesfor a cell population. The set of barcoded binders is incubated with thecell population, resulting in localization of the barcoded binders onthe cell biomarkers (e.g. biomarker#1 on the cell surface is bound byantibody#1, which has a linked oligonucleotide sequence that encodes theidentity of antibody#1). After washing unbound binders, the linkage tothe binder is cleaved (e.g. with a restriction enzyme), and the releasedbarcode is quantified (e.g. by sequencing). The total number of cellsused divided by the total number of antibody#1 barcodes will give theaverage number of biomarker#1 molecules on a cell in the population. Thetotal number of cells used divided by the total number of antibody#2barcodes will give the average number of biomarker#2 molecules on a cellin the population, and so on. As there is no limit to the number ofdifferent types of barcoded binders that can be used to bind to the cellpopulation (other than the number of separate constructions needed ofbarcoded binders), this method has unlimited multiplexing capability.

Compared to FIG. 27A-C, which shows steps for average (i.e., “bulk”)biomarker analysis, FIGS. 28-31 show a scheme for single-cell biomarkercounting. Single-cell biomarker counting can include the same barcodebinder construction, binding, and washing steps as shown in FIG. 27A-C.For single-cell biomarker counting, however, the labeled cells areindividually loaded into droplets, and each droplet is combined with adroplet from a universal barcode droplet library (FIG. 28A-C and FIG.29A-B show cell marker barcodes and universal barcodes of a sticky-endtype, but any suitable type can be used). The cells can be loaded at adilution such that most droplets are empty and the cell-containingdroplets have a single cell. Each droplet that contains a single cellcombines with a droplet that contains a unique barcode, such that all ofthe biomarkers on one cell will have an identical droplet-identifyingbarcode appended, thus enabling later determination of the set ofbiomarkers that were present on the same single cell.

Use of a sticky-ended barcoding droplet library in combination withsample droplets enables collections of individual cells to have theirbiomarkers digitally counted, with different target components in adroplet being labeled with a unique, droplet-identifying barcode,allowing identification and digital quantification of targets present inthe same droplet shown in FIG. 28A-C. The same unlimited multiplexinglevel for cell biomarker analysis is provided for individual cells aswith the bulk averaged cell experiment, and the universal barcodinglibrary can be scaled to have enough binding barcodes to uniquely labela very large number of single cells for digital biomarker analysis.Ligation of the target-identifying and droplet-identifying barcodes intoa composite barcode, as shown in FIG. 29A-B, is followed by processingto read both sequence identity and counts of each composite barcode type(e.g. by sequencing, digital PCR, or microarray detection).

The individual steps for this workflow are shown in FIGS. 28A-31D. FIG.28, panel A shows the preparation of sticky-ended barcoded binders.Panel B shows binding barcoded binders to cell population. Cells can bewashed to remove unbound barcoded binders (Panel C) and then loaded intoa microfluidic device to generate single cell droplets (Panel D) (e.g.,loaded at such a dilution that cell-containing droplets primarilycontain single cells according to Poisson statistics). In FIG. 29, PanelE shows sample droplets being combined with the universal barcodingdroplet library (combination is achieved by droplet interdigitationfollowed by dielectrophoretic pair-wise coalescence). Panel F in FIG. 30releasing the barcoded cells into a single aqueous phase (e.g., lysis bya chemical droplet destabilizer). Cells are washed to remove unboundmaterial (Panel G). Ligase and ATP are added to ligate the annealedcomposite barcodes (alternatively, the ligase and ATP can be included inthe droplet for in-droplet ligation), as shown in Panel H. In FIG. 31,Panel I shows releasing the barcodes by cleavage (e.g. restrictionenzyme). According to Panel J, information from the ligated compositebarcode can be read (e.g. NextGen sequencer). Note that the barcodemembers shown in Figure J can be optionally ligated back together intoconstructs of any length before sequencing according to any knownmethod. Sequencing reveals the type and number of each barcode present.The type and number of each barcode corresponds to the type and numberof biomarkers present on each individual cell. Cell#1-associatedbiomarkers are identified by the 5-prime binder barcode (e.g.biomarker#1 is bound by binder#1 that contains barcode#1 ligated to cellidentifier barcode#1a: 1b) and the number of each type of biomarker onthat cell matches the number of barcodes with all three sequences (i.e.1:1a:1b). In this example, Cell#1 has 1 molecule of biomarker#1, 3molecules of biomarker#2, and 1 molecule of biomarker#n; Cell#2 has 2molecules of biomarker#1, 2 molecules of biomarker#2, and 1 molecule ofbiomarker#n.

Note that in the foregoing, a cleavable linker is included in barcodedbinders (Panel A in FIG. 27A-C); barcode products are ligated together(Panel H, FIG. 30); and the cleavable linkers are subsequently released(Panel I, FIG. 31). The invention provides methods for linking andreleasing oligos. Methods of cleaving and releasing are not limited tothe examples shown in FIGS. 27-30, and may be used generally in methodsof the invention.

FIG. 32A-C illustrates methods for linking and for releasing barcodes.FIG. 32, panel A, shows coupling barcodes by photoligation and releasingbarcodes by photocleavage. FIG. 32, panel B, illustrates includingrestriction sites or binder-specific loops for cleavage enzymes forsubsequent cleavage of barcodes (or for blocking of enzymes).

Barcodes with dPCR

The invention generally provides methods for labeling target materialincluding providing copies of a construct that includes a unique N-merand a functional N-mer. In some embodiments, constructs of the inventionare further analyzed in combination with digital PCR.

For example, sticky-ended Barcodes containing dPCR optimized countersequences can be provided for use in counting barcoded digital sandwichassays. The optimized multiplexing dPCR set can be used in conjunctionwith dELISA panels of any “plexity” including, for example,moderate-plex (e.g. 15-100 plex for just FAM/VIC probes) or higher-plexfor any dELISA panel known in the art (e.g. cytokine panel; viralantigen panel; bacterial antigen panel).

In some embodiments, barcodes can be used with digital PCR for acopy-number variant (CNV) analysis. Methods include generating asticky-ended barcode set that will hybridize to a set of targets thathave disease-associated copy number variation. Using the non-amplifiedversion of the priming universal barcode library on purified DNA willresult in digitally countable (e.g. by optimized multiplex dPCR) CNVanalysis. If a diverse collection of these sticky-ended barcoded primingprobe pairs are provided in a droplet (i.e. each library droplet has thesame barcode, but a mixture of targeting probes) this collection can becombined with genetic material (e.g., a genome, transcriptome, achromosome, a target nucleic acid). The constructs are allowed tohybridize to the target, and then detected or counted according to themethods described herein. From these results, variations in copy numbercan be ascertained.

Barcoded Binders

FIG. 33A-B depicts barcoding binders generally (e.g., for digitalproteomics). Binders are shown in a form generally representing anantibody for purposes of illustration, but methods of barcoding bindersare not labeled to antibodies and include any known binder. As shown inPanel A, a barcode is attached to a binder, optionally via a cleavablelinker and adaptor end. The barcoded binder can be furtherfunctionalized by including an optional universal barcode librarybinding end. The binder shown in a droplet on the right side of Panel Acan also be provided with a binder that is capture-tagged (for example,with biotin) for subsequent selective capture of target material.

Panel B illustrates an optional additional step for adding additionalbarcode information to the barcoded binder. A sticky-end barcode ismerged into the droplet with ligase, the sticky ends hybridize, and theconstruct is ligated together. This produces a binder linked to abarcode including barcode information from two supplied barcodes.

As shown in Panel C, this step can optionally be repeated-any number oftimes—to produce barcodes of increasing complexity, or greater numbersof unique barcodes.

While additional barcodes are shown here being added with ligase, thisis optional. Barcodes can be hybridized on without ligation. Additionalbarcodes can optionally be added with strand-displacement polymerase ortransposase. Suitable binders include, without limit, antibodies,aptamers, nucleic acids, proteins, cofactors, and other bindingmolecules discussed elsewhere herein.

FIG. 34A-B shows an approach to high-plex barcoding of binders. Stepsshown in FIG. 34A-B are generally followed, although multiple bindersare provided. In a first step (e.g., Panel A of FIG. 34A-B), differentbinders are first individually barcoded. The second barcoding step(e.g., Panel B) then adds a droplet-specific barcode to the multiplebinders.

Barcode Sandwich Assays

In certain embodiments, an ELISA sandwich can be formed, for example, bycombining a serum droplet with a droplet containing ELISA reagents(e.g., with one antibody immobilized on a bead and the other insolution). FIG. 35A-B shows a general schematic for a sandwich assayusing barcoded binders.

This aspect of the invention involves several components, such ascreating tagged binding reagent droplet libraries consisting ofindividual members targeting single protein markers, with expandabilityto high levels of multiplexing (e.g. 1000 member library targets 1000proteins/motifs); binding samples to the tagged detecting molecules inpicoliter volume droplets in a highly parallel “single-plex” manner,performing pair-wise combination of droplet reagent libraries and sampledroplets; digital counting of productive binding events after washingand release of a readable DNA sequence tag.

Although the instant invention can utilize any species suitable forbinding to a protein or protein fragment (e.g. aptamers, affibodies), inone example provided herein, the binding species include antibodyreagents. The sandwich principle will be identical (described forantibodies), using appropriate concentrations of two antibodies bindingto different epitopes of the same target antigen for co-encapsulation inthe reagent droplet. One of the antibodies can be biotinylated(available from many commercial sources, or can be made using a numberof kits) and the other can be covalently tagged with a syntheticoligonucleotide. The oligonucleotide tags will be synthesized asamine-linked oligos by a commercial manufacturer (e.g. Sigma Genosys,IDT) and can include restriction enzyme motifs if desired. Wellestablished methods for performing the oligo-tagging of antibodies areused.

Sandwich antibody pairs for many biologically and therapeuticallyrelevant (e.g., cancer) targets are readily available. In one example,well characterized pairs are selected that bind proteins and proteinmotifs that are important in common cancer signaling pathways (usingcell lysate samples), or which have been identified as relevant clinicalbiomarkers (using serum samples). Initial candidates for target proteinsin cell lysate analysis include: Akt and phosphorylated AKT, EGFR andphosphorylated EGFR, Src and phosphorylated Src and TNFRI/II. Initialtargets in serum include, but are not limited to: PSA, soluble TNFRI/II,soluble RANKL, CEA, AFP, CA125, beta2 microglobin.

Automated equipment for generation of primer droplet libraries isdescribed above. This equipment can be used to generate a number oftypes of droplet libraries, including viral particles, antibodies, andbeads. For the binding reagent library, a standard microtiter plate isprepared containing each mixed pair of sandwich reagents at theappropriate concentration as input to the library generation process.

Library reagent droplets of two types are prepared, with or without astreptavidin (SA) capture bead. The binding reagent droplet library ismixed and aliquoted for use in assay development. In preferredembodiments, a droplet library is stable and can be stored for longerthan a year (e.g., 4° C.).

After pair-wise combination of the sample droplets and thetagged-antibody library droplets, and incubation (e.g., FIG. 37A),productive sandwiches containing all target molecules (and uncomplexedbiotinylated antibody) bound to an immobilized streptavidin-containingsurface (either SA beads co-encapsulated with the library reagents, orpost release from the droplets). Washing the binding surface removesunbound material, including all background from non-targeted proteins.Finally, the remaining tags can be released by any means known in theart (e.g., by denaturation or restriction enzyme digestion) for digitalquantification.

Any known method known in the art may be utilized to read the tags. Someexamples include, quantitative PCR with standard equipment, digital PCRtechniques or a tag readout scheme based on NextGen sequencing,including the use of barcoding strategies, and microarrays

FIG. 35A-B shows the use of sticky-end barcodes with sticky-end barcodedbinders for labeling a sample in a sandwich assay. To couple samplebarcode information to a barcode sandwich assay as shown in FIG. 35A-B,a sticky-ended barcode is added to bulk Sample A, then made intodroplets (Step A). In step B, sample droplets are combined with bindershaving sticky-ended barcodes and incubated to form a sandwich centeredaround an analyte. The binder barcode then anneals to the samplebarcode. Ligation can be performed before or after release of sandwichfrom droplet. The droplet contents are released and the full sandwich onthe capture surface is washed. The combined barcode is released (e.g.restriction digest) as shown in panel D. Then, reads for each barcodeare counted (e.g. using sequencing).

FIG. 36A-B shows the use of a universal barcode droplet library in asingle-cell lysate sandwich assays. In the embodiment shown in FIG.36A-B, a bind-and-ligate version of a library is generally illustrated.As shown in FIG. 36A-B, the universal barcoding droplet library can becombined with barcoded binders that are used in droplet-based sandwichassays, enabling very high-plex digital sandwich assays, includingsingle cell lysate sandwich assays.

Cleavable sticky-ended barcoded binders are constructed (e.g., see FIG.34A-B) and combined with matching binders that contain a ‘capture-tag’(see FIG. 34A-B, right hand side) for use as paired binding and captureagents. Each binding pair targets one biomarker or complex, with eachbinder directed to a different epitope of the same biomarker or complexcomponent. The Universal Barcode Droplet Library can be used to generatea very high number of uniquely barcoded binders, by successive additionof sticky-ended barcodes to the previously barcoded binders (two roundsof additional sticky-ended barcode addition are shown in FIG. 34A-B). Byleaving a sticky-end available with the last barcode addition before usewith a sample, a sticky-ended barcode that is provided along with thesample to be targeted by the binding pair can be annealed to thebarcoded binder and subsequently ligated (see FIG. 36A-B). Associationof the sample identifier with the barcoded binder pair identifierresults in a combined barcode, that if captured and read, will identifyboth the binding agent pair and the sample source for the specificanalyte targeted by the binding pair. As an example, in the case wherethe sample droplet contains a lysed single cell, the combination of thesample identifier and the unique binding pair barcode will allowdetermination of co-localization of the target molecules coming from thesame cell lysate after the droplet contents are released, captured on asolid support and washed, and released and read (see FIG. 36A-B).

FIG. 36A-B shows an example workflow for quantifying analytes in asample using barcoded binders in a sandwich assay. As shown in Panel A,two binding reagents types are constructed: barcoded binders andcapture-tag binders. If the barcoded binder has a sticky-end, successivecombination with a universal barcode droplet library enables buildingvery high levels of barcode complexity, such that the number of barcodesexceeds the number of analytes or analyte droplets. Pairs oftarget-specific binders are made into a droplet library (Panel B), witheach set of target binders in separate droplets. As shown in Panel C, asticky-ended sample barcode identifier is added to the sample and sampledroplets are generated, and (Panel D) combined with the library dropletsto initiate highly parallel ‘single-plex’ binding reactions. Afterbinding is complete, productive sandwiches are captured via thecapture-tag (streptavidin (SA) biotin (B) interaction shown in Panel F),and washed to remove unbound material. The captured barcodes arereleased, recovered, and processed for reading as shown in Panel G.Reads for each barcode are counted (e.g. using sequencing).

FIG. 37A-J shows a number of examples of single or multiple targetbarcode sandwich assays. Panel A shows a binder pair targeting twodifferent regions of the same analyte enable counting single targetanalytes. As shown in Panels B and C, binder pairs targeting differentanalytes in a complex enable identification and digital quantificationof analyte complexes. Panel D shows a binder pair targeting twodifferent regions of the same analyte, with one target being a specificmodification (e.g. protein post-translational). Panel E showscross-linked or stable complexes that can be analyzed (e.g.protein-nucleic acid). Panels F-J show the identification and countingof various nucleic acid molecules and motifs. Note that the binderbarcode information includes details on which binders are in the librarydroplet (e.g. “3:1:2” in example C means Binder3 in the same droplet asBinder1 and Capture-Tag Binder2).

Sequencing Barcodes

Having labeled the DNA, RNA, or protein of cell-free material, acollection of cells, single cell, or portion thereof, using the methodsdescribed herein, the labeled (and possibly amplified) sample may besequenced. Sequencing can be carried out using any suitable sequencingtechnique. A particularly useful method for nucleic acid sequencing isone wherein nucleotides are added successively to a free 3′ hydroxylgroup, resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. The nature of the nucleotide added may be determined aftereach nucleotide addition or at the end of the sequencing process.Sequencing techniques using sequencing by ligation, wherein not everycontiguous base is sequenced, and techniques such as massively parallelsignature sequencing (MPSS) where bases are removed from, rather thanadded to the strands on the surface are also within the scope of theinvention.

The invention also encompasses methods of sequencing amplified nucleicacids generated by solid-phase amplification. Thus, the inventionprovides a method of nucleic acid sequencing comprising amplifying apool of nucleic acid templates using solid-phase amplification andcarrying out a nucleic acid sequencing reaction to determine thesequence of the whole or a part of at least one amplified nucleic acidstrand produced in the solid-phase amplification reaction. Theinitiation point for the sequencing reaction may be provided byannealing of a sequencing primer to a product of a solid-phaseamplification reaction. In this connection, one or both of the adaptorsadded during formation of the template library may include a nucleotidesequence which permits annealing of a sequencing primer to amplifiedproducts derived by whole genome or solid-phase amplification of thetemplate library.

The products of solid-phase amplification reactions wherein both forwardand reverse amplification primers are covalently immobilized on thesolid surface are so-called bridged structures formed by annealing ofpairs of immobilized polynucleotide strands and immobilizedcomplementary strands, both strands being attached to the solid supportat the 5′ end. Arrays comprised of such bridged structures provideinefficient templates for typical nucleic acid sequencing techniques,since hybridization of a conventional sequencing primer to one of theimmobilized strands is not favored compared to annealing of this strandto its immobilized complementary strand under standard conditions forhybridization.

In order to provide more suitable templates for nucleic acid sequencing,it may be advantageous to remove or displace substantially all or atleast a portion of one of the immobilized strands in the bridgedstructure in order to generate a template which is at least partiallysingle-stranded. The portion of the template which is single-strandedwill thus be available for hybridization to a sequencing primer. Theprocess of removing all or a portion of one immobilized strand in a‘bridged’ double-stranded nucleic acid structure may be referred toherein as linearization, and is described in further detail in U.S. Pub.2009/0118128, the contents of which are incorporated herein by referencein their entirety.

Bridged template structures may be linearized by cleavage of one or bothstrands with a restriction endonuclease or by cleavage of one strandwith a nicking endonuclease. Other methods of cleavage can be used as analternative to restriction enzymes or nicking enzymes, including interalia chemical cleavage (e.g. cleavage of a diol linkage with periodate),cleavage of abasic sites by cleavage with endonuclease (for example‘USER’, as supplied by NEB, part number M5505S), or by exposure to heator alkali, cleavage of ribonucleotides incorporated into amplificationproducts otherwise comprised of deoxyribonucleotides, photochemicalcleavage or cleavage of a peptide linker.

Following the cleavage step, regardless of the method used for cleavage,the product of the cleavage reaction may be subjected to denaturingconditions in order to remove the portion(s) of the cleaved strand(s)that are not attached to the solid support. Suitable denaturingconditions, for example sodium hydroxide solution, formamide solution orheat, will be apparent to the skilled reader with reference to standardmolecular biology protocols (Sambrook et al., supra; Ausubel et al.supra). Denaturation results in the production of a sequencing templatewhich is partially or substantially single-stranded. A sequencingreaction may then be initiated by hybridization of a sequencing primerto the single-stranded portion of the template.

Thus, the invention encompasses methods wherein the nucleic acidsequencing reaction comprises hybridizing a sequencing primer to asingle-stranded region of a linearized amplification product,sequentially incorporating one or more nucleotides into a polynucleotidestrand complementary to the region of amplified template strand to besequenced, identifying the base present in one or more of theincorporated nucleotide(s) and thereby determining the sequence of aregion of the template strand.

One sequencing method which can be used in accordance with the inventionrelies on the use of modified nucleotides having removable 3′ blocks,for example as described in WO04018497, US 2007/0166705A1 and U.S. Pat.No. 7,057,026, the contents of which are incorporated herein byreference in their entirety. Once the modified nucleotide has beenincorporated into the growing polynucleotide chain complementary to theregion of the template being sequenced there is no free 3′-OH groupavailable to direct further sequence extension and therefore thepolymerase can not add further nucleotides. Once the nature of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides, it ispossible to deduce the DNA sequence of the DNA template. Such reactionscan be done in a single experiment if each of the modified nucleotideshas a different label attached thereto, known to correspond to theparticular base, to facilitate discrimination between the bases addedduring each incorporation step. Alternatively, a separate reaction maybe carried out containing each of the modified nucleotides separately.

The modified nucleotides may be labeled (e.g., fluorescent label) fordetection. Each nucleotide type may thus carry a different fluorescentlabel, for example, as described in U.S. Pub. 2010/0009353, the contentsof which are incorporated herein by reference in their entirety. Thedetectable label need not, however, be a fluorescent label. Any labelcan be used which allows the detection of an incorporated nucleotide.

One method for detecting fluorescently labeled nucleotides comprisesusing laser light of a wavelength specific for the labeled nucleotides,or the use of other suitable sources of illumination. The fluorescencefrom the label on the nucleotide may be detected by a CCD camera orother suitable detection means. Suitable instrumentation for recordingimages of clustered arrays is described in WO07123744 and U.S. Pub.2010/0111768, the contents of which are incorporated herein by referencein their entirety.

In all cases, regardless of the incorporation of molecular barcodes orthe location of the barcodes in the event that they are incorporated,sequencing adaptors can be attached to the nucleic acid product in abi-directional way such that in the same sequencing run there will besequencing reads from both the 5′ and 3′ end of the target sequence. Insome cases it is advantage to use the location of the barcode on the 5′or 3′ end of the target sequence to indicate the direction of the read.It is well known to one skilled in the art how to attach the sequencingadaptors using techniques such as PCR or ligation.

The invention is not intended to be limited to use of the sequencingmethod outlined above, as essentially any sequencing methodology whichrelies on successive incorporation of nucleotides into a polynucleotidechain can be used. Suitable alternative techniques include, for example,the genome sequencers from Roche/454 Life Sciences (Margulies et al.(2005) Nature, 437:376-380; U.S. Pat. Nos. 6,274,320; 6,258,568;6,210,891), the SOLiD system from Life Technologies Applied Biosystems(Grand Island, N.Y.), the HELISCOPE system from Helicos Biosciences(Cambridge, Mass.) (see, e.g., U.S. Pub. 2007/0070349), and the Ionsequencers from Life Technologies Ion Torrent, Ion Torrent Systems, Inc.(Guilford, Conn.).

High Accuracy NGS

Methods of the invention can be used for highly accurate nucleic acidsequencing, particularly by enabling the discrimination between trueSNPs and sequencing errors. This is particularly valuable where anallele frequency may be much lower than 50% (e.g., 1%, 0.01%, etc.), forexample, when testing for loss of heterzygosity in a tumor. The presenceof low percentage mutants in cancer samples due to the heterogeneity ofthe tumor or the presence of normal cells can result in a mutant allelefrequencies below 5%. Other applications such as the detection of cellfree circulating tumor DNA from blood and the detection of minimalresidual disease (MRD) in cancer are other applications requiring thecorrect identification of low percentage alleles in a mixture of otheralleles. Detection of mutations in bacteria or virus that are at a lowpercentage, but which convey resistance to drug therapies, can altertreatment regiments if the potential for resistance is known ahead oftime.

For the applications mentioned above as well as others, there is a needto accurately detect base changes at frequencies 5% or lower. However,this level can be below what is expected for the error rate encounteredthroughout the entire sequencing process. A method is provided that candistinguish between true base changes from those that were introducedthrough error. Methods of the invention include labeling each targetmolecule going into the process with a unique barcode that ends up inthe sequencing read. This allows each sequencing read to be traced backto the original molecule in the sample. This enables the ability todistinguish between base errors and true base changes.

In one illustrative example shown in FIG. 43, a sample has two fragmentsfor the same target region of interest (Target Fragment A and TargetFragment B). In this target region there are two bases of interest (Xand Y). The base at site X is the same in both fragments (C:G). The baseat site Y is different, an A:T in Fragment A and a G:C in Fragment B.The first step is to label each fragment with a unique barcode. FragmentA is labeled with barcode 1 (BC1) and Fragment B is labeled with barcode2 (BC2). In addition in this example sequence corresponding to PCRprimers is also added to the fragment so that each of the fragments canbe amplified by PCR (PCR F, PCR R). After barcode labeling, each of thefragments go through one cycle of PCR. Fragment B is replicatedcorrectly, however, there is base change introduced into Fragment A atsite X changing the C to a T in one of the strands (drawn-in oval inFIG. 43). The sample is subjected to another round of PCR generatingfour PCR products for each fragment. If this sample was sent out forsequencing at this point in which both the barcode and the targetsequence were read and associated, BC1 in the results would reveal asequencing error. This is shown in FIG. 44.

The barcoding allows a user to determine whether a detected base changeis real or an artifact of some kind. In this case sequencing without abarcode would identify the base change at site X of a C to a T to be atrue change present at 12.5% (1 out of 8 reads with a T). However,examining the associated barcode would identify that the there are twodifferent bases (C and T) associated with the same barcode. This couldonly happen if the change happened after barcoding and thus is anartifact of the process. However, the base change at site Y is a truechange because all the bases at site Y are the same for each barcode(BC1 all A, BC2 all G). The attachment of a barcode to DNA or RNA priorto processing allows a user to distinguish a real base change from asequencing artifact even down to very low percentages.

Padlock Probe Library

In certain embodiments, the invention provides a gap-filling padlockprobe library. A padlock barcode library can have sticky-ends, forexample, to combine with universal barcode library building block.

Padlock probes can be hybridized to target (in either asequence-specific manner, or with universal or random sequences), andpolymerase plus ligase can be used to fill in the gap between probeends. This can produce a circular DNA template including a (optionallybarcoded) copy of a target template for downstream processing.

Restriction Barcoding

In certain embodiments, the invention includes restriction barcoding.FIG. 69A-B shows a workflow for restriction barcoding. Cells or nucleicacid are sequestered in droplets. If cells, these can be lysed. Anyrestriction enzyme or combination of restriction enzymes can beintroduced into the cells.

Any nucleic acid can be provided (e.g., a barcode construct of theinvention) having a restriction site. Nucleic acids can be providedsticky-ended, or can be exposed to restriction enzymes to expose stickyends. Barcode sticky ends can be hybridized to target sticky ends, andoptionally ligated.

In some embodiments, a barcode in provided with a restriction site orassociated sticky end, and a capture tag or moiety such as a bead. Aftertarget is connected to the barcode via the sticky end, functional stepscan exploit the bead or capture moiety (e.g., to isolate target). Thenin subsequent downstream processing, for example, target can be releasedby subsequent restriction digestion.

Sticky-Ended Motif-Probes

In certain embodiments, the invention provides (amplified andnon-amplified versions of) probes having sticky ends of a certain motif.General or specific motifs can be used and include transcription factorbinding sites, TATA-box, telomeric sequences, known promoters, or anyother known motif.

For example, a probe having a TATA-box can be provided (e.g. as asinglet, without a corresponding “downstream/3′” partner, or as part ofa pair. A plurality of such probes can be hybridized to a target sample,and polymerase can synthesize a copy of the target from theprobe-binding positions (e.g., the probe can provide a 3′ free hydroxylgroup to seed polymerization). This can proceed by primer extension(e.g., synthesizing a single copy of each target area) or via anamplification reaction.

In certain embodiments, a barcoded sticky-ended motif probe is bound totelomeres, and the count is correlated to telomere length.

III. Probe-Type Labels

In addition to barcode-based methods discussed above, labeled targetmaterial can be analyzed using digital PCR methods or by counting offluorescent probe labels. Digital PCR is discussed below. Methodsfurther include incorporating labels having a fluorescent or othercolorimetric probe using the methods described herein. In someembodiments, labels are incorporated and amplified material is releasedfrom encapsulation and can be input into a digital PCR reaction tosimultaneously screen for multiple genotypes and/or mutations for aplurality of target genes in the sample.

Ideally, the sensitivity of digital PCR is limited only by the number ofindependent amplifications that can be analyzed, which has motivated thedevelopment of several ultra-high throughput miniaturized methodsallowing millions of single molecule PCR reactions to be performed inparallel (discussed in detail elsewhere). In a preferred embodiment ofthe invention, digital PCR is performed in aqueous droplets separated byoil using a microfluidics system. In another preferred embodiment, theoil is a fluorinated oil such as the Fluorinert oils (3M). In a stillmore preferred embodiment the fluorinated oil contains a surfactant,such as PFPE-PEG-PFPE triblock copolymer, to stabilize the dropletsagainst coalescence during the amplification step or at any point wherethey contact each other. Microfluidic approaches allow the rapidgeneration of large numbers (e.g. 10⁶ or greater) of very uniformlysized droplets that function as picoliter volume reaction vessels (seereviews of droplet-based microfluidics). But as will be described, theinvention is not limited to dPCR performed in water-in-oil emulsions,but rather is general to all methods of reaction compartmentalizationfor dPCR. In the description that follows, the invention is described interms of the use of droplets for compartmentalization, but it isunderstood that this choice of description is not limiting for theinvention, and that all of the methods of the invention are compatiblewith all other methods of reaction compartmentalization for dPCR. In yetanother embodiment, the labeled, amplified genetic mixture is analyzedusing an array (e.g., microarray) readout.

Methods of the invention involve novel strategies for performingmultiple different amplification reactions on the same samplesimultaneously to quantify the abundance of multiple different DNAtargets, commonly known to those familiar with the art as“multiplexing”. Methods of the invention for multiplexing dPCR assayspromise greater plexity—the number of simultaneous reactions—thanpossible with existing qPCR or dPCR techniques. It is based on thesingular nature of amplifications at terminal or limiting dilution thatarises because most often only a single target allele is ever present inany one droplet even when multiple primers/probes targeting differentalleles are present. This alleviates the complications that otherwiseplague simultaneous competing reactions, such as varying arrival timeinto the exponential stage and unintended interactions between primers.

In one aspect, the invention provides materials and methods forimproving amplicon yield while maintaining the quality of droplet-baseddigital PCR. More specifically, the invention provides dropletscontaining a single nucleic acid template and multiplexed PCR primersand methods for detecting a plurality of targets in a biological sampleby forming such droplets and amplifying the nucleic acid templates usingdroplet-based digital PCR.

Reactions within microfluidic droplets yield very uniform fluorescenceintensity at the end point, and ultimately the intensity depends on theefficiency of probe hydrolysis. Thus, in another aspect of the methodsof the invention, different reactions with different efficiencies can bediscriminated on the basis of end point fluorescence intensity aloneeven if they have the same color. Furthermore, in another method of theinvention, the efficiencies can be tuned simply by adjusting the probeconcentration, resulting in an easy-to-use and general purpose methodfor multiplexing. In one demonstration of the invention, a 5-plexTaqMan® dPCR assay worked “right out of the box”, in contrast to lengthyoptimizations that typify qPCR multiplexing to this degree. In anotheraspect of the invention, adding multiple colors increases the number ofpossible reactions geometrically, rather than linearly as with qPCR,because individual reactions can be labeled with multiple fluorophores.As an example, two fluorophores (VIC and FAM) were used to distinguishfive different reactions in one implementation of the invention.

Detection

In certain embodiments, after amplification, droplets are flowed to adetection module for detection of amplification products. Forembodiments in which the droplets are thermally cycled off-chip, thedroplets require re-injection into either a second fluidic circuit forread-out—that may or may not reside on the same chip as the fluidiccircuit or circuits for droplet generation—or in certain embodiments thedroplets may be re-injected for read-out back into the original fluidiccircuit used for droplet generation. The droplets may be individuallyanalyzed and detected using any methods known in the art, such asdetecting the presence or amount of a reporter.

An apparatus can include optical or electrical detectors or combinationsthereof. Examples of suitable detection apparatuses include opticalwaveguides, microscopes, diodes, light stimulating devices, (e.g.,lasers), photo multiplier tubes, and processors (e.g., computers andsoftware), and combinations thereof, which cooperate to detect a signalrepresentative of a characteristic, marker, or reporter, and todetermine and direct the measurement or the sorting action at a sortingmodule. FIG. 47 shows a detection apparatus according to certainembodiments. Detecting labeled material in droplets is discussed in U.S.Pub. 2008/0014589; U.S. Pub. 2008/0003142, and U.S. Pub. 2010/0137163.

In certain aspects, the droplets of the invention contain a plurality ofdetectable probes that hybridize to amplicons produced in the droplets.Members of the plurality of probes can each include the same detectablelabel, or a different detectable label. The plurality of probes can alsoinclude one or more groups of probes at varying concentration. Thegroups of probes at varying concentrations can include the samedetectable label which vary in intensity, due to varying probeconcentrations.

In a separate embodiment the detection can occur by the scanning ofdroplets confined to a monolayer in a storage device that is transparentto the wavelengths or method or detection. Droplets stored in thisfashion can be scanned either by the movement of the storage device bythe scanner or the movement of the scanner over the storage device.

The invention is not limited to the TaqMan assay, as described above,but rather the invention encompasses the use of all fluorogenic DNAhybridization probes, such as molecular beacons, Solaris probes,scorpion probes, and any other probes that function by sequence specificrecognition of target DNA by hybridization and result in increasedfluorescence on amplification of the target sequence.

Optical Labels

In particular embodiments, the labels incorporated into the DNA or RNAof a single cell, or portion thereof, are optically labeled probes, suchas fluorescently labeled probes that are attached to a primer (or N-mer)that hybridizes to a unique region of the target. Examples offluorescent labels include, but are not limited to, Atto dyes,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; LaJolta Blue; phthalo cyanine; and naphthalo cyanine. Preferredfluorescent labels are FAM and VIC™ (from Applied Biosystems). Labelsother than fluorescent labels are contemplated by the invention,including other optically-detectable labels.

In a particular embodiment, the optical label can be conjugated to anantibody, an siRNA, an aptamer, or a ribozyme specific for target geneor region of interest on the target.

Labels can be used for identification of the library elements of thevarious types of droplet libraries. Libraries can be labeled for uniqueidentification of each library element by any means known in the art.The label can be an optical label, an enzymatic label or a radioactivelabel. The label can be any detectable label, e.g., a protein, a DNAtag, a dye, a quantum dot or a radio frequency identification tag, orcombinations thereof. Preferably the label is an optical label.

The label can be detected by any means known in the art. Preferably, thelabel is detected by fluorescence polarization, fluorescence intensity,fluorescence lifetime, fluorescence energy transfer, pH, ionic content,temperature or combinations thereof. Various labels and means fordetection are described in greater detail herein.

Specifically, after a label is added to each of the various libraryelements, the elements are then encapsulated and each of the dropletscontains a unique label so that the library elements may be identified.In one example, by using various combinations of labels and detectionmethods, it is possible to use two different colors with differentintensities or to use a single color at a different intensity anddifferent florescence anisotropy.

Quality Control

Optical labels are also utilized in quality control in order to ensurethat the droplet libraries are well controlled, and that equal number ofeach library elements are contained within uniform volumes in eachdroplet library. After 120 minutes of mixing, using 8-labels in a96-member library, the average number of droplets is 13,883 for each ofthe library elements.

In some quality control examples, 384-member libraries were preparedwith eight optical labels; typically 5 to 20 micro-liters of eachlibrary element are emulsified into approximately 10 picoliter volumedroplets so there are about 1 million droplets of each library elementand 384 million droplets in the library.

The eight optical labels are a dye at concentrations that increase by afactor of c (where c ranges from about 1.2 to 1.4) from one opticallabel to the next so that the nth optical label has (c)(n−1) the dyeconcentration of the lowest concentration. Optical labels are used withconcentrations between 10 nM and 1 uM. Typically, the range of opticallabel concentrations for one series of labels is 1 order of magnitude(e.g., 10 nM to 100 nM with a multiplier of 1.43 for each increasinglabel concentration). A larger range of droplet label concentrations canalso be used. Further, multiplexed two-color labels can be used as well.

Plates are prepared with 384 separate library elements in separate wellsof the 384-well plates; 8 of which have optical labels. The libraryelements are made into droplets, collected in a vial, (also known as acreaming tower) and mixed for several hours. The mixer works by flippingthe vial over about once every 30 seconds and then allowing the dropletsto rise. Multiple plates can be emulsified and pooled or collectedsequentially into the same vial.

A small fraction of the droplets are taken out of the vial to verify 1)that the droplets are present in the correct predetermined ratio and 2)that the droplets are of uniform size. Typically, 1,000 to 10,000droplets of each library element (0.384 to 3.84 million QC-droplets) areremoved from the vial through a PEEK line in the center opening in thevial cap by positive displacement with a drive oil infused through theside opening in vial cap. The PEEK line takes the droplets into a porton a microfluidic chip at a rate of several thousand droplets/second;for 10 picoliter droplets at a rate of 3000 droplets/s corresponds to atypical infusion rate of roughly 110 micro-liters/hr. Once on chip thedroplets are spaced out by adding oil before they are imaged and passone droplet at a time through a laser excitation spot. Maximumfluorescence intensity data from individual droplets is collected forall of the QC-droplets and histograms are built to show the number ofdroplets within a given fluorescence intensity range. As expected, ifeight of the library elements have optical labels, then there are eightpeaks in the histograms. The increasing concentration factor c=1.38results in uniformly separated peaks across one decade when plotted on alog scale. The relative number of droplets in each peak is used as aquality metric to validate that the libraries were prepared with theexpected relative representation. In this example, the percent variationis determined to be only 2.7% demonstrating that all library elementshave uniform representation.

Image analysis can be utilized to determine and monitor osmotic pressurewithin the droplets. Osmotic pressure (e.g., two member library preparedwith a small difference in buffer concentration) can effect droplets.Specifically, droplets with a lower salt concentration shrink over timeand droplets with higher salt concentration grow over time, untiluniform salt concentrations are achieved.

Image analysis can also be utilized for quality control of the libraryreformatting process. After the various library elements are generated,pooled and mixed, optical labels can be used to verify uniformrepresentation of all library elements. Additionally, image analysis isused to verify uniform volume for all droplets.

Further, image analysis can be used for shelf life testing byquantifying the materials performance. Droplets are stored in vialsunder a variety of conditions to test droplets stability againstdroplet-droplet coalescence events. Conditions tested includetemperature, vibration, presence of air in vials, surfactant type, andsurfactant concentration. A Quality Score of percent coalescence iscalculated by image analysis. Shelf-life for the droplet libraries ofthe present invention exceed 90 days.

Droplet Digital PCR

In certain aspects, the invention provides methods and systems fordroplet digital PCR including high plexity multiplexing.

An exemplary microfluidic system for droplet generation and readout isdepicted in FIG. 48. The microfluidic system for droplet generation andreadout. As shown in FIG. 48a (droplet generation chip), a continuousaqueous phase containing the PCR master mix, primers, and probes, andtemplate DNA is flowed into the fluidic intersection from the left, andthe carrier oil enters from the top and bottom. An emerging bolus ofaqueous liquid is imaged inside the intersection just prior to snappingoff into a discrete 4 pL droplet as the fluidic strain began to exceedthe surface tension of the aqueous liquid. The steady train of dropletsleaving the intersection toward the right is collected off chip as astable emulsion for thermal cycling. FIG. 48b depicts the dropletspacing for readout. Flows are arranged as in FIG. 48a , except insteadof a continuous phase, the emulsion from (a) is injected from the leftinto the intersection after thermal cycling. The oil drains from theemulsion during off-chip handling, hence the emulsion appears tightlypacked in the image before the intersection. The oil introduced in theintersection separates the droplets and the fluorescence of each dropletis measured at the location marked by the arrow. FIG. 48c depicts acartoon of droplet readout by fluorescence. The relatively infrequentPCR(+) droplets (light gray) flow along with the majority of PCR(−)droplets (dark gray) toward the detector. The droplets are interrogatedsequentially by laser induced fluorescence while passing through thedetection region.

In a serial dilution the average number of target DNA molecules perdroplet-called the “occupancy” from this point forward-decreases indirect proportion to the DNA concentration. The occupancy is calculatedfrom Poisson statistics using the following equation well known to thoseexperienced in the art:

$\begin{matrix}{{{occupancy} = {\ln \left( \frac{P + N}{N} \right)}},} & (1)\end{matrix}$

where P and N are the numbers of PCR(+) and PCR(−) dropletsrespectively.

Digital PCR performance in the emulsion format is validated by measuringa serial dilution of a reference gene, branched chain keto aciddehydrogenase E1 (BCKDHA). Mixtures of the PCR master mix, 1× primersand probe for BCKDHA, and varying concentrations of a mixture of humangenomic DNA (1:1 NA14091 and NA13705) are compartmentalized into overone million 5.3 pL droplets in a water-in-fluorinated oil emulsion usingthe droplet generation microfluidic chip. The emulsion is thermallycycled off-chip and afterwards the fluorescence of each droplet isanalyzed by fluorescence in the readout chip (see FIG. 48).

Droplets are analyzed by fluorescence while flowing through the readoutchip to count the numbers of PCR(+) and PCR(−) droplets (see FIG. 48c ).As each droplet passes the detection zone (marked with an arrow in FIG.48b ), a burst of fluorescence is observed. To account for smallrun-to-run differences in the fluorescence intensity that can occur dueto different chip positioning, etc., each set of data is scaled suchthat the average fluorescence intensity of the empty droplets is 0.1 V.

FIG. 49a shows droplet fluorescence during readout for the mostconcentrated sample. Each discrete burst of fluorescence corresponds toan individual droplet. Two different groups of droplets are evident:PCR(+) droplets peaking at ˜0.8 V and PCR(−) droplets at ˜0.1 V; FIG.49b shows a histogram of the peak fluorescence intensities of dropletsfrom the complete data trace in 6a. PCR(+) and PCR(−) droplets appear astwo very distinct populations centered at 0.78 and 0.10 V, respectively;FIG. 49c shows the serial dilution of template DNA. Open circles:measured occupancies; solid line: the best fit to Eqn 2 (A=0.15, ƒ=4.8,R²-0.9999).

FIG. 49a shows a very short duration of a typical trace of fluorescencebursts from individual droplets for the sample with the highest DNAconcentration in the series. PCR(+) and PCR(−) droplets are easilydiscriminated by fluorescence intensity. The two large bursts offluorescence peaking at ˜0.8 V arose from the PCR(+) droplets, whereasthe smaller bursts due to incomplete fluorescence quenching in thePCR(−) droplets peaked at ˜0.1 V. A histogram of peak intensities fromthe complete data set reveals two clear populations centered at 0.10 and0.78 V (FIG. 49b ), demonstrating that the trend evident in the shorttrace in FIG. 49a is stable over much longer periods of time.Integration over the two populations in FIG. 49b yields a total of197,507 PCR(+) and 1,240,126 PCR(−) droplets. Hence the occupancy is0.15 for this sample by Eqn. 1, corresponding to the expected occupancyof 0.18 based on the measured DNA concentration of 110 ng/μL. Theoccupancy is measured for each sample in the serial dilution and fit tothe dilution equation:

$\begin{matrix}{{{{occupancy}(n)} = \frac{A}{f^{n}}},} & (2)\end{matrix}$

where n is the number of dilutions, A is the occupancy at the startingconcentration (n=0), and ƒ is the dilution factor. The linear fit is inexcellent agreement with the data, with an R² value of 0.9999 and thefitted dilution factor of 4.8 in close agreement with the expected valueof 5.0.

Multiplexing Primers in Digital PCR

Droplet-based digital PCR technology uses a single primer pair perdroplet. This library droplet is merged with a template droplet whichcontains all the PCR reagents including genomic DNA except for theprimers. After merging of the template and the primer library dropletsthe new droplet now contains all the reagents necessary to perform PCR.The droplet is then thermocycled to produce amplicons. In oneembodiment, the template DNA is diluted in the template mix such that onaverage there is less than one haploid genome per droplet. Droplet-baseddigital PCR is described in U.S. Pat. No. 7,041,481; U.S. Pub.2008/0014589; U.S. Pub. 2008/0003142; and U.S. Pub. 2010/0137163, thecontents of each of which are incorporated by reference herein in theirentireties.

Having only one haploid genome (i.e., one allele) per droplet givesdroplet PCR advantages over standard singleplex or multiplex PCR intubes or microwells. For example, in traditional PCR, both alleles arepresent in the reaction mix so if there is a difference in the PCRefficiency between alleles, the allele with the highest efficiency willbe over represented. Additionally, there can be variances in thesequence to which the PCR primers hybridize, despite careful primerdesign. A variance in the primer hybridization sequence can cause thatprimer to have a lower efficiency for hybridization for the allele thathas the variance compared to the allele that has the wild type sequence.This can also cause one allele to be amplified preferentially over theother allele if both alleles are present in the same reaction mix.

These issues are avoided in droplet-based PCR because there is only onetemplate molecule per droplet, and thus one allele per droplet. Thus,even if primer variance exists that reduces the PCR efficiency for oneallele, there is no competition between alleles because the alleles areseparated and thus uniformly amplified.

Because droplet-based digital PCR utilizes only one template moleculeper droplet, even if there are multiple PCR primer pairs present in thedroplet, only one primer pair will be active. Since only one amplicon isbeing generated per droplet, there is no competition between amplicons,resulting in uniform amplicon yield between different amplicons.

A certain amount of DNA is required to generate either a specificquantity of DNA and/or a specific number of PCR positive droplets toachieve sufficient sequencing coverage per base. Because only apercentage of the droplets are PCR positive, approximately 1 in 3 in thestandard procedure, it takes more DNA to achieve the equivalent PCRyield per template DNA molecule. The number of PCR positive droplets andthus the amplicon yield can be increased by adding more genomic DNA. Forinstance, increasing the amount of genomic DNA twofold while maintainingthe number of droplets constant will double the amplicon yield. Howeverthere is a limit to the amount of genomic DNA that can be added beforethere is a significant chance of having both alleles for a gene in thesame droplet, thereby eliminating the advantage of droplet PCR forovercoming allele specific PCR and resulting in allelic dropout.

One way to allow the input of more genomic DNA is by generating moredroplets to keep the haploid molecules per droplet ratio constant. Forinstance doubling the amount of DNA and doubling the amount of dropletsincreases the amplicon yield by 2× while maintaining the same haploidgenome per droplet ratio. However, while doubling the number of dropletsisn't problematic, increasing the amount of DNA can be challenging tousers that have a limited amount of DNA.

The multiplexing of PCR primers in droplets enables the simultaneousincrease in the number of PCR droplets while keeping the amount of inputDNA the same or lower to generate an equal or greater amplicon yield.This results in an overall increase in the amount of PCR positivedroplets and amplicon yield without the consumption of more DNA.

By way of example, if there is an average of 1 haploid genome per every4 droplets or¹4 of the haploid genome per droplet and one PCR primerpair per droplet, the chances of the correct template being present forthe PCR primer in the droplet is 1 out of 4. However, if there are 2 PCRprimer pairs per droplet, then there is double the chance that therewill be the correct template present in the droplet. This results in 1out of 2 droplets being PCR positive which doubles the amplicon yieldwithout doubling the input DNA. If the number of droplets containing the2× multiplexed primers is doubled and the DNA kept constant, then thenumber of PCR positive droplets drops back to 1 in 4, but the totalnumber of PCR droplets remains the same because the number of dropletshave been doubled. If the multiplexing level in each droplet isincreased to 4× and the input DNA is the same, the chance of the correcttemplate molecule being present in each droplet doubles. This results inthe number of PCR positive droplets being increased to 1 in 2 whichdoubles the amount of amplicon yield without increasing the amount ofinput DNA. Thus, by increasing the multiplexing of PCR primers in eachdroplet and by increasing the number of droplets overall, the ampliconyield can be increased by 4-fold without increasing the amount of inputDNA.

Alternatively, if the amplicon yield is already sufficient, byincreasing the multiplexing level for the PCR primers in each droplet,the amount of input genomic DNA can be dropped without sacrificingamplicon yield. For example if the multiplexing level of the PCR primersgoes from 1× to 2×, the amount of input genomic DNA can be decreased by2× while still maintaining the same overall amplicon yield.

Even though the number of PCR primer pairs per droplet is greater thanone, there is still only one template molecule per droplet and thusthere is only one primer pair per droplet that is being utilized at onetime. This means that the advantages of droplet PCR for eliminating biasfrom either allele specific PCR or competition between differentamplicons is maintained.

An example demonstration of droplet-based amplification and detection ofmultiple target sequences in a single droplet is shown in FIG. 50.Multiple copies of 5 sets of primers (primers for TERT, RNaseP, E1a,SMN1 and SMN2) were encapsulated in a single droplet at variousconcentrations along with the template DNA and the PCR master mix.

FIG. 50A is a schematic representation of a droplet having 5 sets ofprimers for PCR amplification of a template sequence and 5 probes, eachlabeled with a fluorescent dye, that binds specifically to the amplifiedsequences; FIG. 50B is a time trace of fluorescence intensity detectedfrom droplets after PCR amplification; FIG. 50C is a scatter plotshowing clusters representing droplets that contain specific amplifiedsequences (TERT, RNaseP, E1a, SMN1 and SMN2).

Probes that specifically bind to TERT, RNaseP, E1a, SMN1 or SMN2 werealso encapsulated in the droplets containing the primers. Probes forTERT, RNaseP and E1a were labeled with the VIC dye and probes for SMN1and SMN2 were labeled with the FAM dye. The sequences for TERT RNaseP,E1a, SMN1 and SMN2 were amplified by PCR. The PCR was conducted with astandard thermal cycling setting. For example:

-   -   95° C. for 10 min    -   31 cycles        -   92° C. for 15 s        -   60° C. for 60 s

At the end of the PCR, the fluorescence emission from each droplet wasdetermined and plotted on a scattered plot based on its wavelength andintensity. Six clusters, each representing droplets having thecorresponding fluorescence wavelength and intensity were shown. TheTERT, RNaseP and E1a clusters showed the fluorescence of the VIC dye atthree distinct intensities and SMN1 and SMN1 clusters showed thefluorescence of the FAM dye at two distinct intensities (FIG. 50C). Thenumber of droplets, each having sequences selected from TERT, RNaseP,E1a, SMN1 and SMN2, can be determined from the scattered plot.

FIG. 51 and FIG. 52 show another demonstration of droplet-basedamplification and detection of multiple target sequences in a singledroplet. Here, five sets of primers (for TERT, RNaseP, E1a, 815A and815G) were encapsulated in a single droplet at various concentrationsalong with the template DNA, the PCR master mix, and the probes. Thefive different probes TERT, RNaseP, E1a, 815A and 815G were alsoencapsulated in the droplets containing the primers. Probes for TERT and815A were labeled with the VIC dye and probes for 815G were labeled withthe FAM dye. For each of RNaseP and E1a, two probes, one labeled withthe VIC dye and the other labeled with the FAM dye, were encapsulated.

The primer-plus-probe droplets were fused with template droplets. PCRreactions were conducted with the fused droplets to amplify thesequences for TERT, RNaseP, E1a, 815A and 815G. The PCR was conductedwith a standard thermal cycling setting.

At the end of the PCR, the fluorescence emission from each fused dropletwas determined and plotted on a scattered plot based on its wavelengthand intensity. FIG. 52 shows six clusters, each representing dropletshaving the corresponding fluorescence wavelength and intensity. The TERTand 815A clusters showed the fluorescence of the VIC dye at two distinctintensities; the 815G clusters showed the fluorescence of the FAM dye;and the RNaseP and E1a clusters showed the fluorescence of both the FAMand the VIC dye at distinct intensities (FIG. 52). The number ofdroplets, each having one or more sequences selected from TERT, RNaseP,E1a, 815A and 815G, can be determined from the scattered plot. The copynumber of RNaseP, E1a, 815A and 815G in the template were determined bythe ratio between the number of droplets having the RNaseP, E1a, 815Aand/or 815G sequences and the number of droplets having the TERTsequence (FIGS. 51-52). FIG. 52B is a table showing the copy number ofspecific sequences shown in FIG. 49B.

In yet another exemplary demonstration of multiplexed primer pairs in adroplet-based digital PCR reaction, two droplet libraries weregenerated: droplet library A was generated where each droplet containedonly one primer pair; and droplet library B was generated where theprimer pairs were multiplexed at 5× level in each droplet. HapMap sampleNA18858 was processed in duplicate with droplet libraries A or B usingstandard procedures. Two ag sample DNA was used for droplet library Aand one ag sample DNA was used for the 5× multiplex droplet library B.After PCR amplification, both droplet libraries were broken and purifiedover a Qiagen MinElute column and then run on an Agilent Bioanalyzer.Samples were sequenced by Illumina on the Illumina GAII with 50nucleotide reads and the sequencing results were analyzed using thestandard sequencing metrics. The results from the 5× multiplexed dropletlibrary B were compared to the singleplex droplet library A as shown inFIG. 45.

The results obtained from the 5× multiplexed droplet library B wereequivalent or better than what was obtained from droplet library A. Themultiplexing of primers delivers the same sequencing results for basecoverage, specificity and uniformity that the singleplexing does withthe added advantage of reduced input DNA as shown in FIG. 45.

In FIG. 45, the following entries appear:

Total reads: total number of sequencing read found within the providedsample data.Mapped reads (%): percentage of total reads that mapped to the humangenome.Specificity: percentage of mapped reads that include the target. Thetarget includes all amplicon sequences with primer sequences excluded.Mean base coverage: average base coverage within the target. The targetincludes all amplicon sequences with primer sequences excluded.C1: % of target that has at least 1× base coverage. Note: non-uniquesequencing reads are mapped randomly.C20: % of target that has at least 20× base coverage.C100: % of target that has at least 100× base coverage.Base coverage (0.2× of mean): % of target that has at least 20% of meanbase coverage.

Monochromatic Copy Number Assay

Traditional digital PCR methods involve the use of a single labeledprobe specific for an individual target. FIG. 53A-E is a schematicdepicting one-color detection of a target sequence using droplet-baseddigital PCR. As shown in Panel A of FIG. 53A-E, a template DNA isamplified with a forward primer (F1) and a reverse primer (R1). Probe(P1) labeled with a fluorophore of color 1 binds to the target geneticsequence (target 1). Droplets are made of diluted solution of templateDNA under conditions of limiting or terminal dilution. Dropletscontaining the target sequence emit fluorescence and are detected bylaser (Panels B and C). The number of microcapsules either containing ornot containing the target sequence is shown in a histogram (D) andquantified (E).

FIG. 54A-D is a schematic depicting two-color detection of two geneticsequences with a microfluidic device. As shown in Panel A of FIG. 54A-D,a template DNA is amplified with two sets of primers: forward primer(F1) and a reverse primer (R1), and forward primer (F2) and a reverseprimer (R2). Probe (P1) labeled with a fluorophore of color 1 binds tothe target 1 and probe (P2) labeled with a fluorophore of color 2 bindsto the target 2 (Panels B and C). Droplets are made of diluted solutionof template DNA under conditions of limiting or terminal dilution.Droplets containing the target sequence 1 or 2 emit fluorescence ofcolor 1 or 2 respectively and are optically detected by laser (Panels Band C). The number of microcapsules containing target 1 or 2 is shown byhistogram in Panel D.

Methods of the invention involve performing accurate quantitation ofmultiple different DNA targets by dPCR using probes with the samefluorophore. FIG. 55A-D is a schematic depicting two-color detection ofthree genetic sequences with a microfluidic device. As shown in Panel Aof FIG. 55A-D, a template DNA is amplified with three sets of primers:forward primers (F1, F2 and F3) and reverse primers (R1, R2 and R3).Probes (P1, P2 and P3) are labeled with fluorophores (color 1, color 2and color 1) and bind to the target genetic sequences (target 1, target2 and target 3) (Panels B and C). Droplets are made of diluted solutionof template DNA under conditions of limiting or terminal dilution.Droplets containing target sequence 1 or 3 emit fluorescence of color 1at two different intensities; and droplets containing target sequence 2emit fluorescence of color 2. The number of droplets containing target1, 2 or 3 is shown by histogram in Panel D.

Recent results from the droplet digital PCR (dPCR) shows that multipleindependent PCR reactions can be run and separately quantified using thesame fluorophore. Specifically, an SMN2 assay yields an unexpectedpopulation of droplets with slightly elevated signal in the FAMdetection channel.

The results are depicted in FIG. 56, which shows two dot plots depictingclusters of genetic sequences detected through fluorescence intensity.The left panel is a dot plot showing four clusters, where SMN1 blockerwas present, each corresponding to droplet containing: topleft—reference sequence (SMARCC1); bottom left-no sequence; bottommiddle—SMN1; and bottom right—SMN2. Right panel is a dot plot showingfour clusters. No block for SMN1 sequence was present. Top left:droplets containing the reference sequence (SMARCC1); bottom left:droplets not containing any sequence; bottom middle: droplets containingsequence for SMN1; and bottom right: droplets containing sequence forSMN2. The shift of the bottom middle cluster in right panel as comparedto left panel confirms that fluorescence intensity provides a verysensitive measurement for the presence of a sequence.

Without intending to be bound by any theory, the simplest explanation isthat the cluster arises from weak association of the SMN2 probe to theSMN1 gene despite the presence of a blocker to that gene (anon-fluorescent complementary probe to the SMN1 gene).

One confirmation of SMN1 as the source of the unexpected cluster was anobserved dependence of the intensity of this feature on the presence ofthe SMN1 blocker. A shift toward higher FAM fluorescent intensities wasobserved in the absence of the blocker (FIG. 56). In another definitiveconfirmation the ratio of the SMN1 (putative) population size to thereference size of 0.96 in perfect agreement with expectation (two copiesof each) (S_131 sample). Another sample, S_122, with the same number ofSMN1 copies yielded a ratio of 0.88 in one run and 0.93 in another, alsoconsistent with the proposed explanation of the unexpected cluster.

Without intending to be bound by any theory, these observations indicatethat SMN2 probe binding to SMN1 DNA yields an elevated fluorescentsignal. A simple kinetic model explaining this phenomenon assumes thatthe hybridization of the SMN2 probe to the SMN1 DNA achieves equilibriumat a faster rate than the polymerase fills in the complementary strand.The amount of probe fluorophore that is released in each thermal cycleis therefore proportional to (or even equal to) the number of boundprobes. Thus the lower the binding affinity the fewer the number ofprobe fluorophores that are released. Due to SMN1 sequence mismatch(es)with the SMN2 probe, the affinity of the probe is certainly expected tobe lower to SMN1 than SMN2. This model also explains the signaldependence on the sMN1 blocker: the blocker competitively inhibits theSMN2 probe hydrolysis by the polymerase exonuclease activity.

It may also be, however, that the probe hybridization does not reachequilibrium before exonuclease activity. In this case, the associationrates would play a more dominant role. Similar logic applies. Thebinding rate to the matching site is likely to be faster than to themismatch site, and the blocker would act to decelerate probe binding tothe mismatch site. The binding of SMN2 probe to SMN1 DNA might bedetectable by conventional bulk qPCR, especially in absence of SMN2, buthighly quantitative results like those shown here are very unlikely.Definitely, there is no report of qPCR or any other techniquequantifying two different DNA sequence motifs with the same colorfluorophore. Sequestration of the individual reactions by singlemolecule amplification within droplets eliminates any confusionregarding mixed contributions to the signal.

The advantage of quantifying DNA with multiple probes of the same colorfluorophore goes beyond two highly homologous sequences, as shown here.Rather, any plurality of sequences of any degree of similarity ordissimilarity can be quantified so long as the different probes havesignificantly different binding occupancies to their respective DNAbinding sites.

Another advantage of dPCR for multiplexed reactions is that thedifferent reactions do not compete with each other for reagents as theywould in qPCR. However, the possibility for unintended cross-reactivityremains. A multiplexed assay can require a more dilute sample. Forinstance, at 10% occupancy a duplex reaction would have double occupancy1% of the time. Hence 1 in 10 PCR+ droplets would be doubles, resultingin a final intensity at least as high and possibly higher than thebrighter of the two probes. For a simple duplex system the contributionfrom each probe could be recovered. In this example the total number ofPCR+ droplets for probe 1 would be (Probe 1)+(Probe1+Probe2).

Higher degrees of multiplexing would require greater dilution. Forexample, for a 4-plex at 1% occupancy the probability of one probeoverlapping any of the other 3 is ˜3%, and that error may be too highfor some applications. The need for large dilutions strongly favors thelarge number of dPCR reactions.

In another example of the invention, a single fluorophore (FAM) was usedin a gene copy number assay for both the reference and the target DNA. Amodel system was used with varying concentrations of plasmid DNA torepresent a change in the target gene copy number, relative to areference gene, equivalent to 0-16 copies of the target gene per cell.BCKDHA and SMN2 plasmid DNA served as the reference and target with 1×and 0.5× primers and probes respectively. With a starting ratio of 8:1SMN2 to BCKDHA, the sample was diluted serially by 2× into a solution ofBCKDHA at the same concentration to vary just the amount of SMN2. Theresultant samples were emulsified, thermally cycled, and over 10⁵droplets were analyzed for each sample as described in the previoussection. The process was repeated in triplicate.

Methods of the invention also include analytical techniques foridentification of fluorescence signatures unique to each probe. FIGS.57a-57b depict histograms of a duplex gene copy number assay using onlyone type of fluorophore by digital PCR.

In this example of the invention, histograms of the droplet fluorescenceintensities are shown in FIG. 57a for three different template DNAsamples: a no template control (dotted line), BCKDHA only (solid line),and 1:1 BCKDHA to SMN2 (dashed line). For clarity, the histograms areshown both overlapped to highlight the similarity for certain peaks, andoffset from each other to reveal all of the features. In the case of 1:1BCKDHA to SMN2, three populations were readily apparent: a dominantfeature appeared at 0.08 V, and two smaller peaks were evident at 0.27and 0.71 V. The dominant feature at 0.08 V was assigned to PCR(−)droplets since both small peaks disappeared, but the large one remained,in the no template control. The peak at 0.71 V was assigned to BCKDHAsince it was the sole feature arising with the addition of just BCKDHA,and the peak at 0.27 V appeared on subsequent addition of SMN2,completing the assignments. A very small peak appeared at ˜0.9 V, notvisible on the scale of FIG. 57a , that corresponded to dropletsoccupied by both genes. As another method of the invention, once thedifferent peaks are identified, droplets within each peak were countedcorresponding to each possible state (PCR(+) for either BCKDHA or SMN2,or both, or PCR(−)), and the gene copy number was then determined fromthe ratio of occupancies. Gene copy numbers for each sample in theserial dilution are plotted in FIG. 57b against expected values(observed ratios of SMN2 to BCKDHA to expected ratios of SMN2 toBSKDHA), with an excellent linear fit (y=1.01x) across the full range(R²=0.9997, slope=1.01), demonstrating accurate and precise measurementof the equivalent of 0 to 16 copies of SMN2 per cell.

It is possible to determine if a heterogeneous sample containedcomponents with different copy level numbers. If the copy numbervariants to be assayed were spaced close enough along the chromosome,the DNA from a sample could be fragmented and encapsulated in dropletsat a level of one haploid genomic equivalent or less per droplet. Thedroplet would also contain a TaqMan assay specific for the copy numbervariant. The intensity of the signal in each droplet would depend on thenumber of copy number variants are present for the sample. Counting ofthe number of droplets of different intensities would indicate thingslike how many cells in a particular sample had what level of copy numbervariants.

Splice Variants

In certain embodiments, target material includes alternatively splicedtranscripts, and the invention provides labels for detecting or countingthe splice variant. TaqMan assays can be designed that are specific foreach of the exons in an RNA transcript. After the RNA is turned intocDNA it can be encapsulated into a droplet at 1 copy or less perdroplet. The droplet would also contain the multiplexed TaqMan assay foreach of the exons. Each of the TaqMan assays would contain a differentprobe but all the probes would have the same fluorescent dye attached.The droplets would be thermocycled to generate signal for each of theTaqMan assays. If there are multiple splice variants in the sample theyeach will contain a different number of exons depending on the splicingevents. The fluorescent intensity of each droplet would be differentdepending on the number of exons present. By counting the number ofdroplets with different intensities it would be possible to identify thepresence and abundance of different splice variants in a sample.

Tuning Probe Intensity

Identifying probes by fluorescence intensity often requires adjustingthe brightness of the probes, particularly for higher-plex assays withdense probe patterns. In the previous section the probes for the genecopy number assay yielded very well resolved peaks (FIG. 57a ). Clearlyroom exists to accommodate one or multiple extra probes in the copynumber assay within the resolution of the measurement, but a method foradjusting the fluorescence intensity of the new probes is required toavoid interference with the existing assay. One method of the inventioninvolves varying the probe and primer concentrations together as a verysimple technique to optimize relative intensities in higher-plexreactions.

FIG. 58A-C is a schematic for tuning the intensity of a detectable labelto a particular target with a microfluidic device. As shown in Panel Aof FIG. 58A-C, a template DNA is amplified with two sets of primers:forward primers (F1 and F2) and reverse primers (R1 and R2). Probes (P1and P2) are labeled with fluorophore of color 1 and bind to target 1 andtarget 2 respectively. Fluorescence from target 2 is lower in intensitythan that from target 1 due to single base mismatch between P2 andtarget 2. As shown in Panel B, template DNA is amplified with two setsof primers: forward primers (F1 and F2) and reverse primers (R1 and R2)(Panel B). Fluorescence from target 2 is lower in intensity than thatfrom target 1 due to the presence of a competing probe 2 that is notlabeled with the fluorophore. As shown in Panel C, template DNA isamplified with two sets of primers: forward primers (F1 and F2) andreverse primers (R1 and R2). Probes (P1 and P2) are labeled withfluorophore of color 1 and bind to target 1 and target 2 respectively.Fluorescence from target 2 is lower in intensity than that from target 1due to the presence of a competing probe 2 that is labeled with adifferent fluorophore.

FIG. 59 shows probe fluorescence intensities throughout a serialdilution of the probes and primers for a different reference gene,ribonuclease P (RNaseP), against a constant amount of genomic DNA fromthe Coriell cell line NA3814 at an occupancy of 0.02 target DNAmolecules per droplet. The probe fluorescent intensities varied indirect proportion to probe concentration over a narrow concentrationrange spanning ˜0.15 to 0.4 aM (R²=0.995)-roughly centered about thetypical probe concentration of 0.2 aM-after compensation for dilutionerrors and other run-to-run differences such as optical realignmentsusing the intensity of the PCR(−) droplets as a reference. In summary,probe intensities can be varied by dilution over a small but adequaterange for the purpose of tuning multiplexed assays without affecting theamplification itself.

Although the example above for adjusting probe fluorescence intensitiesinvolves varying probe and primer concentrations together by the samefactor, the invention is not limited to this method alone for varyingprobe intensity. Other methods include varying just the probeconcentration; varying just the primer concentrations; varying just theforward primer concentration; varying just the reverse primerconcentration; varying the probe, forward, and reverse primersconcentrations in any way; varying the thermal cycling program; varyingthe PCR master mix; incorporating into the assay some fraction of probesthat lack fluorophores; or incorporating into the assay anyhybridization-based competitive inhibitors to probe binding, such asblocking oligomer nucleotides, peptide nucleic acids, and locked nucleicacids. The invention incorporates such methods by themselves or in anycombination.

Higher-Plex Reactions

One method of the invention involves performing higher-plex assays witha single probe color (e.g., fluorophore). As described above, probefluorescent intensities can be adjusted by a variety of means such thateach intensity level uniquely identifies a DNA target. For example,targets T1, T2, T3, and T4 might be uniquely identified by intensitylevels I1, I2, I3, and I4. Not intending to be bound by theory, themaximum number of intensity levels possible for unique identification oftargets is related to the resolution of the different intensitylevels—that is the spread of intensities for each particular probecompared to the separation between the average intensities of theprobes—and it is also related to the intensity of the empty dropletsthat tends to grow with increasing numbers of probes. The number ofintensity levels can be 0, 1, 2, 3, 4, 10, 20, 50, or any number (e.g.,up to 100, or higher). In the examples show below, as many as threeintensity levels are demonstrated.

Another method of the invention involves performing higher-plex assaysusing multiple different probe colors (i.e. fluorophores). As above forthe monochromatic multiplexing assay, for each color probe, multipletargets can be identified based on intensity. Additionally, multiplecolors that are spectrally separable can be used simultaneously. Forexample, a single droplet might contain four different probes formeasuring four different targets. Two probes might be of color A withdifferent intensities (say, A1 and A2), and the other two probes ofcolor B with different intensities (say B1 and B2). The correspondingtargets are T1, T2, T3, and T4 for A1, A2, B1, and B2 respectively. If adroplet shows an increase in fluoresce in color A, the droplet thereforecontained either targets T1 or T2. Then, based on the fluorescenceintensity of color A, the target could be identified as T1 or the targetcould be identified as T2. If, however, a droplet shows an increase influorescence in color B, the droplet therefore contained either targetsT3 or T4. Then, based on the fluorescence intensity of color B, thetarget could be identified as T3 or the target could be identified asT4. Not intending to be bound by theory, the maximum number of differentcolors possible is limited by spectral overlap between fluorescenceemission of the different fluorophores. The maximum number of colors canbe 1, or 2, or 3, or 4, or up to 10, or up to 20. The maximum number ofcolors can be higher than 20. In the demonstrations that follow, thelargest number of colors is two.

Another method of the invention involves performing higher-plex assaysusing multiple different probe colors (i.e. fluorophores), howeverunlike the strategy above where each target is identified by single typeof probe with a unique color and intensity, instead in this method asingle target may be identified by multiple probes that constitute aunique signature of both colors and intensities. For example, a singledroplet might contain four different probes for measuring threedifferent targets (say, T1, T2, and T3). Two probes might be of color A(say, A1, and A2), and two probes might be of color B (say, B1 and B2).T1 is measured by probe A1, T2 is measured by probe B1, but T3 ismeasured by both probes A2 and B2. Thus, when a droplet contains T1 onlyincreased fluorescence appears in color A. When a droplet contains T2only increased fluorescence appears in color B. However when a dropletcontains T3, increased fluorescence appears in both colors A and B.

Generally, without wishing to be constrained by theory, the above threemethods for higher-plex dPCR are simplest to implement under conditionsof terminal dilution, that is when the probability of multiple differenttarget molecules co-occupying the same droplet is very low compared tothe probability of any single target occupying a droplet. With multipleoccupancy arises the complexity of simultaneous assays competing withinthe same reaction droplet, and also complexity of assigning theresulting fluorescence intensity that involves a combination offluorescence from two different reaction products that may or may not beequal to the sum of the two fluorescence intensities of the individualreaction products. However, methods of the invention can accommodatethese complications arising from multiple occupancy.

Methods of the invention for higher-plex reactions also include methodsfor primer and probe pairing. In the simplest case targets are unlikelyto reside on the same DNA fragments, such as when targets are fromdifferent cells; or when targets are from different chromosomes within asingle cell type; or when targets are distant from each other within asingle chromosome such that they become physically separated during DNAfragmentation; or when targets are very close to each other within achromosome, but nevertheless become separated by targeted cleavage ofthe DNA, such as by restriction enzyme digestion; or for any otherreason. In such cases each probe can be paired with a single set ofprimers (forward and reverse). However, in other cases the targetregions might frequently reside on the same DNA fragments, for examplewhen targets reside within the same codon, or for any other reason. Insuch cases, a single set of primers might serve for multiple probes (foran example, see Pekin et al.).

Higher multiplex reactions can be performed to distinguish thehaplotypes of two SNPs. For example, assume that at position one therecan be genotypes A or A′ and at position two there can be genotypes of Bor B′. In a diploid genome four unique haplotypes are possible (A,B;A,B′; A′,B; and A′,B′). If for example A′ and B′ represent drugresistant mutations for infection, it is often the case that A′B and AB′are less severe and treated differently than A′B′ which represents asignificant drug resistance that must be treated with extreme care.Digital PCR with intensity discrimination is ideally suited foridentifying low prevalence of A′B′ in a background of mixtures of theother three haplotypes. Haplotyping information is also important forconstruction of haplotypes (e.g., human leukocyte antigen (HLA)haplotypes). One way that the present example can be constructed is byassay design such that color one is used for A and is of high or lowintensity indicative of allele A or A′ respectively and color two isused for B and is of high or low intensity respectively indicative of Bor B′. Populations of [color1,color2] corresponding to [Low, Low] wouldbe a measure of an allele of AB and [high, low] allele A′B and an alleleof [A′B′] will be readily distinguishable as [high, high] even in abackground that is predominately a mixture of A′B and AB′. See FIG. 68.In some cases it will be advantageous to start by encapsulating into thedroplets long single molecules of nucleic acid that contain both A and BSNP location and in other cases it will be desirable to start byencapsulating single cells, bacteria or other organism within thedroplets prior to releasing the nucleic acid from the organism. In stillother embodiments the multiplex intensity detection of multiplesimultaneous targets can be used as surrogate markers for multiple typesof binding interactions or labeling of target materials. This techniqueis also not limited to single molecule detection and can be used forhaplotype detection in single cells (e.g., bacteria, somatic cells,etc.). In single cell analysis, a sorting step may be applied prior tohaplotyping.

5-Plex SMA Assay

The invention in general provides multiplex assays for genetic markers.Here is discussed a 5-plex assay for spinal muscular atrophy (SMA). Theinvention includes other “plex” levels and other genetic markers. SMAwas selected for one example due to both its important clinicalsignificance as well as its complicated genetics. It is the second-mostprevalent fatal neurodegenerative disease and affects ˜1 in 10,000 livebirths. SMA is most often caused by homozygous absence of exon 7 withinthe survival of motor neuron 1 gene (SMN1, reviewed by Wirth et al.),however the severity of the condition is modulated by the number of genecopies of SMN2 with prognosis ranging from lethal to asymptomatic over1-5 copy numbers (reviewed by Elsheikh et al.). Hence accuratequantitation of SMN2 copy number is important for clinical prognosis andgenetic counseling. Aside from large deletions of SMN1, a number ofsingle point mutations or short deletions/duplications within the samegene also account for ˜4% of cases of SMA. In a significant step towarda comprehensive SMA assay, the multiplexed dPCR assay demonstrated herecontains both copy number assays (for SMN1 & 2) and an assay for one ofthe prevalent SNPs (c.815A>G).

One embodiment of the invention is a multi-plex assay for diagnostics.Here, a 5-plex assay quantifies common genetic variants impacting SMAincluding two copy number assays for the SMN1 and SMN2 genes with BCKDHAas a reference, and a SNP assay for the c.815A>G mutation. Twodifferently colored fluorophores, FAM and VIC, were used to uniquelyidentify each of the assays. The probes for SMN1 and SMN2 contained onlyFAM, and for c.815A only VIC. However, mixtures of VIC and FAM-labeledprobes were used for BCKDHA and c.815G. The use of VIC and FAMfluorophores in this example does not limit the invention, rather the5-plex assay can be used with any suitable hybridization-based probechemistries. For validating the assay, a model chromosome wassynthesized containing a single target region for each of the differentprimer/probe pairs. EcoRV restriction sites flanked each target,allowing separation of the fragments.

As another method of the invention, histogram-based data presentationand analysis is incorporated into the invention for identifying andcharacterizing statistically similar populations of droplets that arisefrom one probe signature (color and intensity), and for discriminatingone population of droplets from the others. FIG. 60a shows a2-dimensional histogram of droplet fluorescence intensities as acontoured heat map, with hotter colors representing higher occurrences.Standard techniques were used to compensate for spectral overlap of theFAM and VIC signals. Samples were run at 0.006 occupancy per target. Sixpopulations were clearly evident, five for the assay and one for PCR(−)droplets. As one method of the invention, the populations were assignedby selective exclusion of assay components. For example, excluding theSMN2 primers and probe eliminated the population at the bottom right inthe histogram, but otherwise the distribution remained unchanged.

FIG. 60a is a 2-D histogram of droplet fluorescence intensities, shownas a heat map, for the 5-plex assay against the synthetic modelchromosome for validation. The six well resolved droplet populationscorresponded to the five individual assays plus the empty droplets; FIG.60b shows the results of the SMA pilot study.

Assignments are labeled in FIG. 60a . As we have found to be generallytrue for this method of multiplexing, the assay worked immediately withwell resolved or at least distinguishable populations for each target.As another method of the invention, the relative positions of thedifferent populations in the histogram were then adjusted into aregularly spaced rectangular array by tuning the probe concentration asdescribed in the previous section. Usually no more than two iterationsare required for optimization.

In another method of the invention, the different populations weresufficiently well resolved to allow droplets within each population tobe counted by integration across rectangular boundaries. The boundarieswere positioned at mid-sections between neighboring peaks. The methodsof the invention are not constrained to rectangular boundaries, or tospecific boundary locations between peaks. Rather, any closed orunclosed boundary condition can suffice. Boundary conditions do not needto be “binary” either, in the sense that weighted integrations can alsobe performed across the boundaries to arrive at droplet counts. The peakposition of each cluster varied by no more than 2% from run to run afternormalization to the intensity of the empty droplets to account forvariations in detection efficiency (data not shown). Hence, onceidentified, the same boundaries for integration could be reused betweensamples. The methods of the invention are not limited to fixed boundarypositions. Dynamic population identification and boundary selection inbetween samples or studies is anticipated. Twenty different patientsamples from the Coriell cell repositories were analyzed with thisassay: 4 afflicted with SMA, 1 SMA carrier, and 15 negative controls.Assay results are shown in FIG. 60b . Gene copy number was calculated asbefore, as the ratio of occupancies derived from the number of targetdroplets vs. reference droplets. Like the copy number measurement inFIG. 57, each assay yielded ratios very close to the expected integervalues, but when all of the patient data was plotted as actual ratio vs.expected integer ratio a small systematic deviation from the ideal slopeof 1 was observed. Measured slopes were 0.92, 0.92, and 0.99 for SMN1,SMN2, and c.815A respectively. For clarity, the data in FIG. 60b wasscaled to the ideal slope of 1.

The measured genotypes of the different patients were consistent withtheir disease conditions (unafflicted, carrier, or afflicted). Thepatients afflicted with SMA each had zero copies of SMN1 (numbers SMA1-4 in FIG. 60b ), the carrier had just one copy, and the negativecontrols all had two or three copies (numbers 1-15). Three unrelatedindividuals (numbers 6, 8, and 9) had three copies of SMN1, occurring ata rate of 20% which is similar to a previous report for healthyindividuals. Variability in SMN1 copy number is not surprising since itlies within an unstable region of chromosome 5q13. A larger variety ofSMN2 copy numbers was observed. One to two copies were most common inthe control group, although one individual had zero copies, adistribution consistent with expectations for normal individuals. TheSMA carrier and afflicted patients had elevated copy numbers of SMN2 onaverage: 5 for the carrier, two afflicted with 3 copies, and the otherswith 2 copies. The afflicted patients were all diagnosed as SMA Type I,the most severe form, based on clinical observations according to theCoriell repository. The strong genotype/phenotype correlation betweenSMN2 copy number and disease severity suggests that the two individualswith three copies of SMN2 might have an improved Type II prognosis,especially for the patient SMA 1 who had survived to three years at thetime of sampling, much beyond the typical maximum life expectancy forSMA Type I of 2 years. However there remains reluctance to predictdisease outcome based on SMN2 copies alone since other less wellcharacterized or unknown modifying genes may impact prognosis andbecause not all SMN2 copies may be complete genes. Furthermore some TypeI patients have begun surviving longer in newer clinical settings.Hence, with little clinical information regarding the patients availableto us, we can conclude that our SMN2 assay results were consistent withbroad expectations for disease severity.

The SNP assay revealed that all patients carried the normal c.815Agenotype and no instances of c.815G were observed. The mutation isrelatively rare and hence was not expected to appear in a small patientpanel. Of interest, however, was the presence of an apparent extra genefragment in two unrelated individuals that was uncovered with the SNPassay. The c.815A>G assay does not discriminate between SMN1 and SMN2due to their high sequence similarity, and hence the total copies ofc.815A and G should equal the sum of the copies of SMN1 and SMN2. Thiswas true for all patients except for healthy patients number 1 and 2,both of whom had one extra copy of c.815A. c.815 lies on exon 6, and theSNP that discriminates between the SMN1 and SMN2 genes lies on exon 7,hence the extra genes may be fragments of SMN1 lacking exon 7. Thisseems reasonable because the deletion of exon 7 is the common mutationcausing 95% of cases of SMA (reviewed by Wirth et al.) and it is carriedby 1/40 to 1/60 adults. Thus these patients might have been typicalcarriers of SMA but for the acquisition of at least one compensatinghealthy copy of SMN1 on the same chromosome.

9-Plex SMA Assay

A 9-plex assay for certain SMA related targets was also demonstratedwith just two colors (probes containing FAM and VIC fluorophores). Asidefrom the optimized primer and probe concentrations, assay conditions andexperimental procedures were identical to the 5-plex assay above. FIG.61a shows the various droplet populations in 2-D histograms beforeoptimization of probe concentrations. The identity of the differenttargets is shown on the figure itself. As one method of the invention,the identification of the different populations was made as before, byselective exclusion and/or addition of one or more assays. Most of thepopulations were already well resolved, with the exception of the probefor the c.815A genotype that was in close proximity with the clustercorresponding to empty droplets. After three iterations of optimizationof probe concentrations, all of the target populations were wellresolved from each other, and well resolved from the empty droplets(FIG. 61b ). Three methods of the invention were highlighted in thisdemonstration: (1) nine DNA targets were uniquely identified in atwo-dimensional histogram, far beyond the capabilities of conventionalqPCR; (2) target DNA molecules were distinguished on the basis of somecombination of both color and intensity arising from one or multipleprobes against the same target; and (3) the relative positions of thetarget molecules within the histogram were adjusted by varying the probeconcentrations to optimize the pattern of colors and intensities forincreased resolution amongst the various droplet populations.

As one method of the invention, different droplet populations wereidentified by selective addition or exclusion of assays in the examplesabove. However the invention is not limited to this method alone.Rather, any method for population assignments known to those in the artare considered. Methods of the invention include any method that cancause an identifiable displacement, appearance, or disappearance of oneor more populations within the histograms including changing the probeand primer concentrations together, either by the same factor or bydifferent factors; changing the probe concentration alone; changing theprimer concentrations alone; changing the thermal cycling conditions;and changing the master mix composition. Another method of the inventiontakes advantage of prior knowledge of the position of an assay within ahistogram to assist assignment.

Multiplexing Capacity

The level of multiplexing demonstrated in the preceding SMA example was9×, significantly exceeding the maximum practicable number with qPCR.Without wishing to be constrained by theory, the two main limitationsare the resolution between assays and the increasing fluorescenceintensity of empty droplets with higher loading of probes. A method ofthe invention involves optimizing the pattern of colors and intensitiesof the different probes for maximum multiplexing while still achievingadequate specificity for each individual reaction. Although rectangulararrays of droplet populations were demonstrated for the 5- and 9-plexreactions, another desirable pattern is the tight-packed hexagonalarray. However the invention is not constrained to any particular arraystrategy.

Adding extra colors would increase the capability even further, howeverwith some diminishing returns because the fluorescence of the emptydroplets would continue to rise. The capacity could be yet furtherincreased with better probes yielding larger differential signals, suchas hybrid 5′-nuclease/molecular beacon probes that reduce background bycontact quenching yet exhibit the bright signals typical of freeunquenched fluorophores. With such improvements multiplexing capacityexceeding 50× can be envisioned.

Multiplexing with Optical Labeling

Using droplet-based microfluidics, multiple targets can also be measuredsimultaneously by a different method. According to the alternativemethod, primers and probes can be loaded individually into dropletsalong with an optical label to uniquely identify the assay. Typicallythe optical label is a fluorophore, or a combination of differentfluorophores, that are spectrally distinct from the probe fluorophore.Various different types of droplets, each containing different assaysthat are uniquely identified by different optical labels, can be mixedinto a “library” of droplets. Then, according to methods of theinvention above, library droplets are merged one-to-one with dropletscontaining template DNA. After thermal cycling, some droplets thatcontain template DNA will exhibit brighter fluorescence at the emissionwavelengths of the probes. The specific target DNA molecules giving riseto these PCR(+) signals are subsequently identified by the opticalprobes. In one study, the six common mutations in KRAS codon 12 werescreened in parallel in a single experiment by one-to-one fusion ofdroplets containing genomic DNA with any one of seven different types ofdroplets (a seven-member library), each containing a TaqMan® probespecific for a different KRAS mutation, or wild-type KRAS, and anoptical code.

In one method of the invention, optical labeling can be combined withthe various methods for multiplexing dPCR already incorporated into thisinvention. For example, a single optical label might code for the entire5-plex SMA assay, above, instead of just a single assay as in the KRASexample above. In this manner, other optical labels might code fordifferent screening assays for newborn infants. According to othermethods of the invention, above, a single DNA sample from an infantcould then be analyzed with all of the assays simultaneously by mergingdroplets containing the DNA one-to-one with library droplets containingthe optically encoded assays.

As an example of combining multiplexing with optical labels, a so called3×3×3 combination multiplex reaction with optical labeling wasdemonstrated (3×3 optical labeling with two fluorophores, each encodinga triplex assay, for a total of 27-plex). Two fluorophores were employedfor optical labeling, Alexa633 and CF680 (excited by a 640 nm laser),with three intensity levels each producing nine total optical labels. Asbefore with the 5- and 9-plex assays for SMA, TaqMan assays were usedwith FAM and VIC fluorophores (excited by a 488 nm laser). Thefluorescence from the FAM and VIC fluorophores were recordedsimultaneously with the fluorescence from the optical labels, requiringmodifications to the optical layout of the instrumentation described forthe SMA assay (the optical schematic for two-laser excitation and4-color detection is shown in entirety in FIG. 62). Also, co-flowmicrofluidics were used in this example (the use of co-flow basedmicrofluidics for this application is one of the methods of theinvention described above). In this case, the template DNA wasintroduced into the chip in one flow, and the PCR master mix, theprimers and probes for one triplex assay, and the unique composition offluorophores for the optical label were introduced into the chip inanother flow simultaneously. The two flow streams converged in a fluidicintersection upstream from the droplet forming module, and thus eachdroplet formed contained the contents of both flow streams. Methods toimplement co-flow microfluidics are well known to those in the art. Thedroplets were collected, and then the procedure was repeated with thenext triplex assay and optical label. The procedure was repeated a totalof nine times, once for each pair of assays and optical labels. All ofthe droplets were collected into a single PCR tube and thermally cycledoff chip. The mixture of thermally cycled droplets was reinjected intothe same read-out chip as used for the SMA assay, above, and thefluorescence intensities of the assays from all four fluorophores wasrecorded.

FIG. 63 shows the cumulative results from all droplets in the 3×3×3assay using co-flow microfluidics. The figure shows two 2-D histogramsof droplet fluorescence intensities, the histogram on the left from allof the optical labels, and the histogram on the right from the assays.The contributions from all droplets are shown, that is, from threedifferent triplex assays. (Both panels) 2-D histograms shown as heatmaps with hotter colors representing higher droplet counts. (Left panel)histogram of optical labels, i.e. fluorescence intensities of dropletsmeasured at wavelengths for the two fluorophores comprising the opticallabels. (Right panel) assay histogram, i.e. fluorescence intensities ofdroplets measured at wavelengths suitable for FAM detection (x-axis),and VIC detection (y-axis). Both histograms were compensated forspectral overlap by standard techniques.

Standard methods were used to compensate for spectral overlap. Thehistograms if FIG. 63 are shown as a heat maps, with hotter colorsdesignating larger numbers of droplets. Nine different clusters ofdroplets were clearly evident in the histogram of the optical labels,corresponding to each of the nine different optical labels: there is asmall group of four clusters at the bottom left corner of the histogram,corresponding to optical labels with the lowest fluorescent intensities;and there are five clusters appearing as linear streaks at the higherintensities. The droplet clusters were less distinct in the histogram,but this was as expected because the droplets shown contained all of thetriplex assays. The individual assays became clearly distinct once asingle type of assay was selected by using the optical labels, asfollows.

Methods of the invention involve selecting individual populations ofdroplets all containing the same optical labels, or groups of opticallabels. In some methods of the invention, boundaries of fluorescenceintensity were used to specify populations. In the example shown here, arectangular boundary was used specifying the minimum and maximumfluorescence intensities for each fluorophore. However the methods ofthe invention are not restricted to rectangular boundaries. Anyboundary, closed or unclosed, can be employed. Furthermore, according tomethods of the invention, selections of droplet populations can be madeby any method, and is not restricted to threshold-based methods such asboundary selection.

FIG. 64A shows the droplet fluorescence intensities for the assay (righthistogram) when only one optical label was selected (left histogram).Selections were taken from all of the droplets from FIG. 64A-C. Each ofthe three different selections in panels A-C were for optical labelsencoding the same assay (TERT, SMN1, and SMN2). Histograms are asdescribed in FIG. 63. (Left histograms, optical labels) Superimposedlines demark the bounding box for selecting a single optical label.(Right histograms, assay) Only droplets containing the selected opticallabel are displayed.

The lines overlaid on the histogram of the optical labels identify therectangular boundary used to select just the optical label with thelowest fluorescence for both fluorophores. Both histograms showed onlythe droplets that were selected. After selection, four distinct clustersof droplets appeared in the assay histogram, three for the differentassays (in this case, assays for SMN1, SMN2, and TERT, where TERT isanother common reference gene) and one for the empty droplets. The copynumbers for SMN1 and SMN2 were measured by the same methods of theinvention as described above for the 5-plex SMA assay, with values of1.8 and 0.94 close to the expected values of 2 and 1, respectively. Thesame assay was encoded with two other optical labels, and theirselections are shown in FIGS. 20B and C. Similar results were achieved,with an overall measurement of 1.9±0.1 and 0.9±0.1 copies of SMN1 andSMN2 respectively, showing the measurement to be accurate withinexperimental uncertainty.

FIGS. 65A, B, and C show optical label selections for a different assay(TERT, c.5C in the SMN1 gene, and BCKDHA (labeled E1a in the figure)).Selections were taken from all of the droplets from FIG. 19. Each of thethree different selections in panels A-C were for optical labelsencoding the same assay (TERT, c.5C from SMN1, and BCKDHA). Histogramsare as described in FIG. 63. (Left histograms, optical labels)Superimposed lines demark the bounding box for selecting a singleoptical label. (Right histograms, assay) Only droplets containing theselected optical label are displayed.

In each case shown in FIG. 65A-C four distinct clusters also appeared,and accurate measurements of gene copy number were made for c.5C andBCKDHA, referenced to TERT, of 2.9±0.1 and 2.0±0.2 compared to 3 and 2,respectively.

FIGS. 66, B, and C show optical label selections for a third assay(TERT, c.88G in the SMN1 gene, and RNaseP, where RNaseP is a commonreference gene). Selections were taken from all of the droplets fromFIG. 63. Each of the three different selections in panels A-C were foroptical labels encoding the same assay (TERT, c.88G from SMN1, andBCKDHA). Histograms are as described in FIG. 63. (Left histograms,optical labels) Superimposed lines demark the bounding box for selectinga single optical label. (Right histograms, assay) Only dropletscontaining the selected optical label are displayed. Accurate gene copynumbers of 2.1±0.1 were measured for both c.88G and RNaseP, referencedto TERT, compared to the expected value of 2.

In summary, the demonstration here shows use of nine different opticallabels to enable independent measurement of three triplex assays in asingle experiment. Although some of the optical labels encoded forredundant assays in this example (there were only three different assaysdespite having nine optical labels), the invention is not constrained toany particular formatting of assays and optical labels. Embodiments ofthe invention include formats where all of the assays are the sameacross all of the optical labels; where none of the assays are the sameacross all of the optical labels; where some of the assays are the sameacross all of the optical labels; where some of the assays have greaterplexity than others across all of the optical labels; where all of theassays have the same plexity across all of the optical labels; and anyother arrangements of assays across all of the optical labels areconsidered.

Although two different fluorophores were used to create the opticallabels in this example, the invention is not constrained to anyparticular number of fluorophores comprising the optical labels.Embodiments of the invention include optical labels comprised of 1fluorophore, or 2 fluorophores, or 3 fluorophores, or 4 fluorophores, orup to 10 fluorophores, or up to 20 fluorophores. Optical labels can alsocomprise more than 20 fluorophores. Although solely triplex assays wereused in the example demonstration here, the invention is not constrainedto use of triplex assays with optical labels. Embodiments of theinvention include plexities of the following amounts when used withoptical labels: single plex, duplex, triplex, 4-plex, up to 10-plex, upto 20-plex, up to 50-plex, and up to 100-plex. Embodiments of theinvention also include plexities exceeding 100 when used with opticallabels.

Another method of the invention involves the use of droplet merging,instead of co-flow, for combining multiplexing with optical labels. Ademonstration using droplet merging was performed with the same 3×3×3assay as in the preceding example with co-flow. The assays (probes andprimers) combined with their unique optical labels were firstencapsulated into droplets along with the PCR master mix. Subsequently,according to methods of the invention described above, a librarycontaining a mixture of droplets from all nine optically labeled assayswas merged one-to-one with droplets containing template DNA from thesame patient as in the preceding example. As another method of theinvention, the droplet merge was performed using a lambda-injector stylemerge module, as described in U.S. Provisional Application, Ser. No.61/441,985, incorporated by reference herein. Aside from the differencesbetween co-flow and merge, the assays and experimental procedures wereidentical to those above for the co-flow experiment. FIG. 67A-J shows2-D histograms of droplet fluorescence intensity for the optical labelsand the assays that are similar to those in FIGS. 63-66. FIG. 67A-Jdepicts a dPCR assay combining multiplexing with optical labels usingdroplet merging. As in the case for co-flow, upon selection of dropletscontaining individual optical labels, the expected distinct clusters ofdroplets corresponding to each assay were clearly evident. Furthermorefor each assay the measured gene copy number matched or very nearlymatched the expected values within experimental uncertainty (See FIG.46).

Although methods of the invention include using either microfluidicswith co-flow or droplet merging, the invention is not limited in thisregard. Any fluidic method capable of generating optically labeleddroplets that also contain fluorogenic DNA hybridization probes areconsidered. For example, other embodiments well known in the art aremixing optical labels and assays in the macrofluidic environment beforeinjection into a droplet generating chip; and mixing optical labels andassays thoroughly upstream from the droplet forming module in dedicatedmixing modules, such as with a serpentine mixer.

Data Analysis

One method of the invention involves histogram-based data presentationand analysis for identifying and characterizing populations ofstatistically similar droplets that arise from unique probe signatures(color and intensity), and for discriminating one population of dropletsfrom the others. Another method of the invention involveshistogram-based data presentation and analysis for identifying andselecting populations of droplets based on unique signatures fromoptical labels. Examples of one and two-dimensional histograms have beenprovided for these methods, but the invention is not limited in thisregard. As described above, it is anticipated that greater numbers ofcolors will be used for both multiplexing and for optical labels. Hence,embodiments of the invention include histograms of dimensionalitygreater than two, such as 3, or 4, or up to 10, or up to 20. Histogramsof dimensionality greater than 20 are also incorporated into theinvention.

Another method of the invention involves the selection of dropletswithin histograms, either for counting, or for assay selection as in theuse of optical labels, or for any other purpose. Methods of theinvention include selections by boundaries, either closed or unclosed,of any possible shape and dimension. Methods of the invention alsoinclude selections of droplets that exhibit fluorescence from singletypes of fluorophores, or from multiple types of fluorophores, such asarising from multiple probes against a common DNA target.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1: Oligonucleotide Labeling of Single Cell Genomes

Barcoding of single cell genomes is performed following encapsulation,cell lysis, and temperature-sensitive proteolysis of single cells indroplets (see FIG. 40A-B for overall workflow). Single cell droplets aregenerated using a limited dilution regime loading of a dispersed cellsuspension into a microfluidic droplet generation device (as previouslydescribed). Additional droplet manipulations can also be performed (e.g.droplet sorting) in advance of the step where the droplets are combinedwith the labeling droplet library. After combining the singlegenome-containing droplets with barcoded droplet libraries, and eithermultiple displacement amplification (MDA) enzymes for whole genomeamplification (WGA) (e.g. using the enzyme phi29 (see FIG. 21)),transposase enzymes (see FIG. 22)), or other amplification enzymes andreagents, each cell's genome becomes labeled with a barcode that willidentify the amplified genomic loci as coming from a unique genomeduring subsequent sequencing and analysis. In the example scheme shownin FIG. 21 for MDA WGA, the ‘tailed barcoded primer’ labeling reagent istri-partite, with a universal tail portion (e.g., oligonucleotidesequences for use in sequencing library construction) immediately 5′ toa barcode sequence, followed by one of a set of random hexamer basesthat enable priming from multiple places in the genome. In the examplescheme for using a transposase (FIG. 22), the ‘tailed barcoded primer’labeling reagent is bi-partite, with a universal tail portionimmediately 5′ to a barcode sequence. The labeling enzyme and anyrequired buffer or co-reaction component can either be included in theprimer droplet library or added subsequently via droplet fusion methods.Data regarding the cell lysis and temperature-responsive proteolysissteps are shown in FIG. 41. Data regarding whole genome amplificationusing phi29 in droplets is shown in FIG. 42.

Example 2: Oligonucleotide Labeling of Single Chromosomes

Barcoding of single chromosomes can be performed following encapsulationof individual chromosomes in droplets (see FIG. 43 for overallworkflow). First cells are treated (e.g. nocadazole) to cause metaphasearrest (synchronizing the cell cycle and condensing the chromosomes) andthe cell membranes are lysed (e.g. osmotic pressure) to release thecondensed chromosomes. Limited proteolysis separates the chromosomepairs into individual chromatids. Single-chromosome droplets aregenerated using a limited dilution regime loading of a dispersedchromatid suspension into a microfluidic droplet generation device(previously described). Additional droplet manipulations can also beperformed (e.g. droplet sorting, additional proteolysis using athermo-responsive protease) in advance of the step where the dropletsare combined with the labeling droplet library. After combining thesingle chromosome-containing droplets with barcoded droplet libraries,and either MDA enzymes for WGA (e.g. phi29) (see FIG. 21) or transposaseenzymes (see FIG. 22), each chromosome becomes labeled with a barcodethat will identify the amplified genomic locus as coming from a uniquechromosome during subsequent sequencing and post-process analysis.

Example 3: Oligonucleotide Labeling of Single Cell RNAs

Barcoding of single cell RNAs can be performed following encapsulationand lysis of single cells in droplets (see FIG. 23A-B for overallworkflow). Single cell droplets are first generated using limiteddilution regime loading of a dispersed cell suspension into amicrofluidic droplet generation device (previously described).Additional droplet manipulations can also be performed (e.g. dropletsorting) in advance of the step where the droplets are combined with thelabeling droplet library. One can choose to label and recover severaltypes of RNA collections: polyA-tailed mRNA (using poly dT affinityreagents), sequence-selected subsets of RNA species usingsequence-specific primers, or other subsets (e.g. using random hexamerprimers). The example scheme described here (and shown in FIG. 23A-FIG.26B) is for recovering and labeling the entire polyA+ mRNA compliment ofa cell, i.e. the transcriptome. For this example (FIG. 23A-B), the‘biotinylated tailed barcoded primer’ labeling reagent is tri-partite,with a biotinylated universal tail portion (including a 5′ biotinylatedoligonucleotide sequence that will be used for both capture ontostreptavidin beads and for use in subsequent amplification) immediately5′ to a barcode sequence, followed by a string of T's (poly dT) endingin an ‘anchoring’ sequence (NV; with V degenerate for the 3 bases otherthan T, and N degenerate for all bases). Example workflows are shown inFIG. 23A-B (Flow Chart), FIG. 24 (including optional upfront sorting,and cell lysis within droplets using a temperature-inducible protease),FIG. 40A-B (including optional upfront sorting, and cell lysis withindroplets using a detergent and heat), and FIG. 26A-B (capture beads areincluded in the droplet library). Following combination of the lysedcell droplet with the barcoded mRNA capture primer library, the dropletsare incubated for a time sufficient for binding the mRNA to the primerlibrary, and the resulting hybrids are subsequently released from thedroplet emulsion by addition of a droplet destabilizing reagent. Theaqueous phase containing the mRNA hybridized to the biotinylated captureprimers is incubated with immobilized streptavidin (or a workflow isused that includes capture beads in the primer droplet library, see FIG.40A-B), and the bound complexes are washed in preparation for reversetranscription using the universal tail primer. The resulting materialfrom these procedures is barcoded first strand cDNA, with all of themRNA from each individual cell encoded with the same barcode. Standardsteps for processing cDNA for sequencing are performed, and sequencingof this collection will provide a digital count of each captured mRNAassigned to a barcode that is unique for each cell. The above processcan be conducted on selected RNAs from the transcriptome using theprocedure outline above or using sequence-specific capture primers.

Example 4: Oligonucleotide Labeling of Single DNA Molecules

Haplotype-like information about variation in DNA sequence along acontiguous stretch of DNA is challenging to acquire using current sampleprep and sequencing technologies. In particular, there is a need fordetermining ‘haplotype phasing’ of long stretches of genomic sequencedata derived from ‘short read’ sequencing platforms. Individual singlenucleotide polymorphism (SNP) and collections of SNPs can be determined,but the assignment of a series of SNPs to either of the 2 allelespresent in a diploid genome cannot be performed beyond the ‘read-length’of the sequencing platform (unless individual chromosomes are isolatedfor sequencing). By including barcodes in multiplexed tiled PCRreactions within droplets, this aspect of the invention enables‘haplotype phase’ assignments to be made using current short-readsequencing platforms, and allows this haplotype information to becorrelated with patient's disease propensity, and ultimately to be usedas a genomic biomarker for disease propensity and therapeutic treatment.

As an example, the Illumina sequencing platform generates sequence datawith a read length of 125 bp per amplicon. If paired-end reads are used,one can potentially generate high quality reads 125 bp from both ends ofa 250 bp amplicon. As each 250 bp amplicon is generated from a singlemolecule of target DNA, any number of SNPs identified along thisamplicon are unambiguously ‘in phase’ with each other, allowing a‘haplotype’ to be defined for this 250 bp region. However, it is notcurrently possible to get phased haplotype information across a longertarget DNA stretch using ‘tiled’ amplicons, as sequence from adjacent250 bp amplicons could come from either allele in the sample, and onewould need to know that the tiled amplicons were all generated from thesame DNA template strand.

Several aspects of the invention are combined to enable assignment of aseries of SNPs to target DNA stretches longer than the read-length ofthe sequencing platform, such as a PCR primer library that containsprimers with a large number of barcodes, multiplexed PCR primers thatwill not cross-hybridize with each other and which will uniquely amplifythe target DNA locus, and droplet based amplification. The overallworkflow example is shown in FIG. 35A-B. Optionally, the target locuscan be pre-amplified using a single pair of PCR primers that flanks theentire locus, before appropriate loading of the sample into droplets foramplification and barcoding (not depicted in the workflow in FIG.35A-B).

In an exemplary embodiment, the ‘tailed barcoded primer’ labelingreagent is tri-partite, with a universal tail portion (for use insubsequent amplification) immediately 5′ to a barcode sequence followedby the sequence-specific targeting primer bases. A primer dropletlibrary ‘member’ includes a droplet that contains all of the targetedprimers sufficient for covering the target bases, each with the samebarcode that will enable post-sequencing correlation to the targetstrand. The number of library members is determined by the ratio ofbarcode number to the number of target alleles to be analyzed. By way ofexample, without limitation, FIG. 35A-B shows 100 cells as input, with4000 barcodes giving a 1/10 chance of duplicate barcodes for any allele.In this example, the DNA from 100 cells provides 400 target alleles,which is loaded (together with polymerase, buffer, and nucleotides) intoone million droplets and combined with the barcoding primer library togenerate a PCR-competent droplet emulsion. As an example for a 3 kbtarget region, 13 tiled primer pairs can be used to cover all of thetarget bases. Fewer primer pairs can be used if only subsets of thetarget bases need to be phased. After thermocycling, the amplifiedproducts are released into a single bulk aqueous phase (e.g., using adroplet de-stabilizing reagent), and a subsequent PCR reaction isperformed using the universal primer tail and any sequencingplatform-specific adaptor (and additional barcodes) needed beforesequencing. Examples of the PCR inputs and outputs are shown in FIG.11A-C. Using a yield threshold of ˜150 sequencing reads as being morethan sufficient for high confidence SNP calling, the total number of PCRcycles (droplet PCR plus bulk PCR) can be limited to 10 cycles(sufficient to generate 150 copies).

Example 5: Oligonucleotide Labeling of Digital Sandwich Assays with dPCRReadout

Barcoding of digital sandwich assays is performed using barcoded bindersand capture tagged-binders (e.g. antibody ELISA pair) constructed in asimilar manner as shown in FIG. 33A-B or FIG. 37A-J, constituting asandwich assay barcoded binder library. The overall workflow for thedroplet barcoded sandwich assay is shown in FIG. 70A-B: A) Two bindingreagents types are constructed: Barcoded Binders and Capture-TagBinders; B) Pairs of target-specific binders are made into a dropletlibrary, with each set of target binders in separate droplets; C) Thesample is made into sample droplets, and D) combined with the librarydroplets to initiate highly parallel ‘single-plex’ binding reactions.After binding is complete, productive sandwiches are E) captured via thecapture-tag (e.g. streptavidin (SA) biotin (B) interaction shown), andwashed to remove unbound material; F) The captured barcodes arereleased, recovered, and processed for reading; G) Reads for eachbarcode are counted.

In this example the barcodes added to the barcoded binders areconstituted such that they are targets for dPCR analysis, with optimizedbarcode sequences that enable optically resolved multiplex dPCRanalysis. For example, if an optimized set of 15 barcode targets isconstructed for identification of 15 different target proteins, thencounting the number of each barcode type using the optimized dPCRreadout will enable a 15-plex sandwich assay (e.g. 15 differentcytokines can be quantified from a blood sample after combination withthe Sandwich Assay Barcoded Binder Library and readout using a 16-plexoptimized dPCR scheme with 4 concentrations of FAM and 4 concentrationsof VIC TaqMan probes).

In addition, the dPCR optimized barcodes can be sticky-ended, enablingadditional barcoding information to be added (e.g. a sticky-endedbarcode present in the sample droplet can be ligated to the barcodedbinder, such that the final released barcode has an additional motifthat can include a fluorescent moiety that is optically resolved fromFAM and VIC, enabling higher-plex analysis).

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

1-31. (canceled)
 32. A method for analyzing a nucleic acid, the methodcomprising: obtaining nucleic acid from a cell or a population of cells;exposing the nucleic acid to a transposase to form a reaction product;introducing the reaction product to a droplet comprising barcodedprimers and reagents for amplification; conducting an amplificationreaction in the droplet to produce barcoded amplicons corresponding totransposase-accessible regions of the nucleic acid; and sequencing thetransposase-accessible regions.
 33. The method of claim 32, wherein thenucleic acid comprises genomic DNA.
 34. The method of claim 32, whereinthe exposing step results in reaction products in an aqueous suspension.35. The method of claim 32, wherein the reaction product is a transposednucleic acid.
 36. The method of claim 32, wherein the introducing stepcomprises forming a plurality of droplets, each comprising a differentreaction product.
 37. The method of claim 32, wherein the dropletcomprises a single molecule of the reaction product.
 38. The method ofclaim 32, wherein the droplet is formed by intersecting an aqueous fluidcomprising the reaction product with a counter-propagating stream ofoil.
 39. The method of claim 32, wherein each of the barcoded primerscomprises a functional N-mer portion and a unique N-mer portion.
 40. Themethod of claim 39, wherein all unique N-mers within a droplet areidentical.
 41. The method of claim 32, wherein the barcoded primershybridize at random locations on the reaction product.
 42. The method ofclaim 32, wherein the barcoded primers are attached to beads.
 43. Themethod of claim 32, further comprising breaking the droplets after theconducting step.
 44. The method of claim 43, wherein breaking thedroplets comprises releasing the barcoded amplicons into an aqueousphase.
 45. The method of claim 32, wherein the barcoded amplicons arenot attached to beads.